Prof. Dr. Albert Sickmann

associated member

Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V.


  • A Homozygous PPP1R21 Splice Variant Associated with Severe Developmental Delay, Absence of Speech, and Muscle Weakness Leads to Activated Proteasome Function
    Hentschel, A. and Meyer, N. and Kohlschmidt, N. and Groß, C. and Sickmann, A. and Schara-Schmidt, U. and Förster, F. and Töpf, A. and Christiansen, J. and Horvath, R. and Vorgerd, M. and Thompson, R. and Polavarapu, K. and Lochmüller, H. and Preusse, C. and Hannappel, L. and Schänzer, A. and Grüneboom, A. and Gangfuß, A. and Roos, A.
    Molecular Neurobiology 60 (2023)
    view abstract10.1007/s12035-023-03219-9
  • Multi-omics approaches to study platelet mechanisms
    Solari, F.A. and Krahn, D. and Swieringa, F. and Verhelst, S. and Rassaf, T. and Tasdogan, A. and Zahedi, R.P. and Lorenz, K. and Renné, T. and Heemskerk, J.W.M. and Sickmann, A.
    Current Opinion in Chemical Biology 73 (2023)
    view abstract10.1016/j.cbpa.2022.102253
  • Periostin as a blood biomarker of muscle cell fibrosis, cardiomyopathy and disease severity in myotonic dystrophy type 1
    Nguyen, C.D.L. and Jimenez-Moreno, A.C. and Merker, M. and Bowers, C.J. and Nikolenko, N. and Hentschel, A. and Müntefering, T. and Isham, A. and Ruck, T. and Vorgerd, M. and Dobelmann, V. and Gourdon, G. and Schara-Schmidt, U. and Gangfuss, A. and Schröder, C. and Sickmann, A. and Gross, C. and Gorman, G. and Stenzel, W. and Kollipara, L. and Hathazi, D. and Spendiff, S. and Gagnon, C. and Preusse, C. and Duchesne, E. and Lochmüller, H. and Roos, A.
    Journal of Neurology (2023)
    view abstract10.1007/s00415-023-11633-1
  • Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles
    Mohamud Yusuf, A. and Hagemann, N. and Zhang, X. and Zafar, M. and Hussner, T. and Bromkamp, C. and Martiny, C. and Tertel, T. and Börger, V. and Schumacher, F. and Solari, F.A. and Hasenberg, M. and Kleinschnitz, C. and Doeppner, T.R. and Kleuser, B. and Sickmann, A. and Gunzer, M. and Giebel, B. and Kolesnick, R. and Gulbins, E. and Hermann, D.M.
    Basic research in cardiology 117 (2022)
    Antidepressants have been reported to enhance stroke recovery independent of the presence of depressive symptoms. They have recently been proposed to exert their mood-stabilizing actions by inhibition of acid sphingomyelinase (ASM), which catalyzes the hydrolysis of sphingomyelin to ceramide. Their restorative action post-ischemia/reperfusion (I/R) still had to be defined. Mice subjected to middle cerebral artery occlusion or cerebral microvascular endothelial cells exposed to oxygen-glucose deprivation were treated with vehicle or with the chemically and pharmacologically distinct antidepressants amitriptyline, fluoxetine or desipramine. Brain ASM activity significantly increased post-I/R, in line with elevated ceramide levels in microvessels. ASM inhibition by amitriptyline reduced ceramide levels, and increased microvascular length and branching point density in wildtype, but not sphingomyelinase phosphodiesterase-1 ([Smpd1]-/-) (i.e., ASM-deficient) mice, as assessed by 3D light sheet microscopy. In cell culture, amitriptyline, fluoxetine, and desipramine increased endothelial tube formation, migration, VEGFR2 abundance and VEGF release. This effect was abolished by Smpd1 knockdown. Mechanistically, the promotion of angiogenesis by ASM inhibitors was mediated by small extracellular vesicles (sEVs) released from endothelial cells, which exhibited enhanced uptake in target cells. Proteomic analysis of sEVs revealed that ASM deactivation differentially regulated proteins implicated in protein export, focal adhesion, and extracellular matrix interaction. In vivo, the increased angiogenesis was accompanied by a profound brain remodeling response with increased blood-brain barrier integrity, reduced leukocyte infiltrates and increased neuronal survival. Antidepressive drugs potently boost angiogenesis in an ASM-dependent way. The release of sEVs by ASM inhibitors disclosed an elegant target, via which brain remodeling post-I/R can be amplified. © 2022. The Author(s).
    view abstract10.1007/s00395-022-00950-7
  • Cathepsin D as biomarker in cerebrospinal fluid of nusinersen-treated patients with spinal muscular atrophy
    Schorling, D.C. and Kölbel, H. and Hentschel, A. and Pechmann, A. and Meyer, N. and Wirth, B. and Rombo, R. and Sickmann, A. and Kirschner, J. and Schara-Schmidt, U. and Lochmüller, H. and Roos, A. and Abele, T.B. and Andres, B. and Angelova-Toshkina, D. and Baum, P. and Baum, T. and Baumann, M. and Baumgartner, M. and Baur, U. and Becker, B. and Behring, B. and Bernert, G. and Birsak, T. and Bellut, J. and Bertsche, A. and Blankenburg, M. and Blaschek, A. and Braun, N. and Braun, S. and Burgenmeister, N. and Claus, N. and Cordts, I. and de Vries, H. and Deba, T. and Marina, A.D. and Denecke, J. and Deschauer, M. and Dörnbrack, K. and Driemeyer, J. and Eckenweiler, M. and Eisenkölbl, A. and Fiedler, B. and Fischer, M. and Flotats-Bastardas, M. and Freigang, M. and Friese, J. and Gaiser, P. and Gebert, A. and Geitmann, S. and Goldhahn, K. and Grässl, M. and Gröning, K. and Grosskreutz, J. and Gruber-Sedlmayr, U. and Guillemot, H. and Günther, R. and von der Hagen, M. and Hagenacker, T. and Hahn, A. and Hartmann, H. and Hiebeler, M. and Hobbiebrunken, E. and Friedrich Hoffmann, G. and Holtkamp, B. and Holzwarth, D. and Horber, V. and Husain, R.A. and Illsinger, S. and Jansen, E. and Johannsen, J. and Kaindl, A. and Kaiser, N. and Klamroth, J. and Christoph Koch, J. and Köhler, C. and Koelker, S. and Kolzter, K. and Korschinsky, B. and Küpper, H. and Langer, T. and Lehnert, I. and Lingor, P. and Löscher, W.N. and LoudoviciüKrug, D. and Martakis, K. and Mayer, I. and Metelmann, M. and Meyer, S. and von Moers, A. and Mueller-Kaempffer, K. and Müller, M. and Müller, P. and Müller-Felber, W. and Neuwirth, C. and Niederschweiberer, J. and Nolte, A. and Odorfer, T. and Omran, H. and Pauschek, J. and Pickrodt, K. and Plecko, B. and Pühringer, M. and Quinten, A.L. and Rappold, M. and Reihle, C. and Reinhardt, T. and Rödiger, A. and Roetmann, G. and Saffari, A. and Schimmel, M. and Schlachter, K. and Schneider, J. and Schoene-Bake, C. and Schreiber, G. and Schwartz, O. and Schwerin-Nagel, A. and Schwersenz, I. and Smitka, M. and Stein, S. and Steinbach, R. and Steiner, E. and Steuernagel, D. and Stögmann, E. and Stolte, B. and Stoltenburg, C. and Stüve, B. and Tassoni, A. and Theophil, M. and Thiele, S. and Topakian, R. and Trollmann, R. and Türk, M. and van der Stam, L. and Vill, K. and Vogt, S. and Vollmann, P. and Walter, M.C. and Warken, B. and Weber, M. and Weiler, M. and Weiß, C. and Weiss, D. and Weiss, S. and Wenzel, F. and Wider, S. and Wiebe, N. and Wiegand, G. and Wilichowski, E. and Wilken, B. and Wochner, K. and Zeiner, F. and Zeisler, D. and Zeller, D. and Zemlin, M. and Ziegler, A. and the SMArtCARE consortium
    European Journal of Neurology 29 (2022)
    Background and purpose: The therapeutic landscape of spinal muscular atrophy (SMA) has changed dramatically during the past 4 years, but treatment responses differ remarkably between individuals, and therapeutic decision-making remains challenging, underlining the persistent need for validated biomarkers. Methods: We applied untargeted proteomic analyses to determine biomarkers in cerebrospinal fluid (CSF) samples of SMA patients under treatment with nusinersen. Identified candidate proteins were validated in CSF samples of SMA patients by Western blot and enzyme-linked immunosorbent assay. Furthermore, levels of peripheral neurofilament heavy and light chain were determined. Results: Untargeted proteomic analysis of CSF samples of three SMA type 1 patients revealed the lysosomal protease cathepsin D as a candidate biomarker. Subsequent validation analysis in a larger cohort of 31 pediatric SMA patients (type 1, n = 12; type 2, n = 9; type 3, n = 6; presymptomatically treated, n = 4; age = 0–16 years) revealed a significant decline of cathepsin D levels in SMA patients aged ≥2 months at the start of treatment. Although evident in all older age categories, this decline was only significant in the group of patients who showed a positive motor response. Moreover, downregulation of cathepsin D was evident in muscle biopsies of SMA patients. Conclusions: We identified a decline of cathepsin D levels in CSF samples of SMA patients under nusinersen treatment that was more pronounced in the group of "treatment responders" than in "nonresponders." We believe that our results indicate a suitability of cathepsin D levels as a possible biomarker in SMA also in older patients, in combination with analysis of peripheral neurofilament light chain in adolescents or alone in adult patients. © 2022 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.
    view abstract10.1111/ene.15331
  • Cyclophilin A Is Not Acetylated at Lysine‐82 and Lysine‐125 in Resting and Stimulated Platelets
    Rosa, A. and Butt, E. and Hopper, C.P. and Loroch, S. and Bender, M. and Schulze, H. and Sickmann, A. and Vorlova, S. and Seizer, P. and Heinzmann, D. and Zernecke, A.
    International Journal of Molecular Sciences 23 (2022)
    Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed—neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ijms23031469
  • Effects of Extracellular Vesicles from Osteogenic Differentiated Human BMSCs on Osteogenic and Adipogenic Differentiation Capacity of Naïve Human BMSCs
    Wang, C. and Stöckl, S. and Li, S. and Herrmann, M. and Lukas, C. and Reinders, Y. and Sickmann, A. and Grässel, S.
    Cells 11 (2022)
    Osteoporosis, or steroid-induced osteonecrosis of the hip, is accompanied by increased bone marrow adipogenesis. Such a disorder of adipogenic/osteogenic differentiation, affecting bone-marrow-derived mesenchymal stem cells (BMSCs), contributes to bone loss during aging. Here, we investigated the effects of extracellular vesicles (EVs) isolated from human (h)BMSCs during different stages of osteogenic differentiation on the osteogenic and adipogenic differentiation capacity of naïve (undifferentiated) hBMSCs. We observed that all EV groups increased viability and proliferation capacity and suppressed the apoptosis of naïve hBMSCs. In particular, EVs derived from hBMSCs at late-stage osteogenic differentiation promoted the osteogenic potential of naïve hBMSCs more effectively than EVs derived from naïve hBMSCs (naïve EVs), as indicated by the increased gene expression of COL1A1 and OPN. In contrast, the adipogenic differentiation capacity of naïve hBMSCs was inhibited by treatment with EVs from osteogenic differentiated hBMSCs. Proteomic analysis revealed that osteogenic EVs and naïve EVs contained distinct protein profiles, with pro-osteogenic and anti-adipogenic proteins encapsulated in osteogenic EVs. We speculate that osteogenic EVs could serve as an intercellular communication system between bone- and bone-marrow adipose tissue, for transporting osteogenic factors and thus favoring pro-osteogenic processes. Our data may support the theory of an endocrine circuit with the skeleton functioning as a ductless gland. © 2022 by the authors.
    view abstract10.3390/cells11162491
  • Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells
    Trifunovic, S. and Smiljanić, K. and Sickmann, A. and Solari, F.A. and Kolarevic, S. and Divac Rankov, A. and Ljujic, M.
    Respiratory Research 23 (2022)
    Background: Although still considered a safer alternative to classical cigarettes, growing body of work points to harmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect of e-cigarettes needs to be investigated in more detail considering their widespread use. Methods: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, with and without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cellular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performed via high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry. Results: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however it negatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletion in total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteins were upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of posttranslational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data are available via ProteomeXchange with identifier PXD032071. Conclusions: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMs alterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact. © 2022, The Author(s).
    view abstract10.1186/s12931-022-02102-w
  • Endoplasmic reticulum-stress and unfolded protein response-activation in immune-mediated necrotizing myopathy
    Preusse, C. and Marteau, T. and Fischer, N. and Hentschel, A. and Sickmann, A. and Lang, S. and Schneider, U. and Schara-Schmidt, U. and Meyer, N. and Ruck, T. and Dengler, N.F. and Prudlo, J. and Dudesek, A. and Görl, N. and Allenbach, Y. and Benveniste, O. and Goebel, H.-H. and Dittmayer, C. and Stenzel, W. and Roos, A.
    Brain Pathology 32 (2022)
    Patients suffering from immune-mediated necrotizing myopathies (IMNM) harbor, the pathognomonic myositis-specific auto-antibodies anti-SRP54 or -HMGCR, while about one third of them do not. Activation of chaperone-assisted autophagy was described as being part of the molecular etiology of IMNM. Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)-stress accompanied by activation of the unfolded protein response (UPR) often precedes activation of the protein clearance machinery and represents a cellular defense mechanism toward restoration of proteostasis. Here, we show that ER/SR-stress may be part of the molecular etiology of IMNM. To address this assumption, ER/SR-stress related key players covering the three known branches (PERK-mediated, IRE1-mediated, and ATF6-mediated) were investigated on both, the transcript and the protein levels utilizing 39 muscle biopsy specimens derived from IMNM-patients. Our results demonstrate an activation of all three UPR-branches in IMNM, which most likely precedes the activation of the protein clearance machinery. In detail, we identified increased phosphorylation of PERK and eIF2a along with increased expression and protein abundance of ATF4, all well-documented characteristics for the activation of the UPR. Further, we identified increased general XBP1-level, and elevated XBP1 protein levels. Additionally, our transcript studies revealed an increased ATF6-expression, which was confirmed by immunostaining studies indicating a myonuclear translocation of the cleaved ATF6-form toward the forced transcription of UPR-related chaperones. In accordance with that, our data demonstrate an increase of downstream factors including ER/SR co-chaperones and chaperones (e.g., SIL1) indicating an UPR-activation on a broader level with no significant differences between seropositive and seronegative patients. Taken together, one might assume that UPR-activation within muscle fibers might not only serve to restore protein homeostasis, but also enhance sarcolemmal presentation of proteins crucial for attracting immune cells. Since modulation of ER-stress and UPR via application of chemical chaperones became a promising therapeutic treatment approach, our findings might represent the starting point for new interventional concepts. © 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.
    view abstract10.1111/bpa.13084
  • ERK1/2 Activity Is Critical for the Outcome of Ischemic Stroke
    Schanbacher, C. and Bieber, M. and Reinders, Y. and Cherpokova, D. and Teichert, C. and Nieswandt, B. and Sickmann, A. and Kleinschnitz, C. and Langhauser, F. and Lorenz, K.
    International Journal of Molecular Sciences 23 (2022)
    Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt ) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt ) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ijms23020706
  • Identification of a novel homozygous synthesis of cytochrome c oxidase 2 variant in siblings with early-onset axonal Charcot-Marie-Tooth disease
    Gangfuß, A. and Hentschel, A. and Rademacher, N. and Sickmann, A. and Stüve, B. and Horvath, R. and Gross, C. and Kohlschmidt, N. and Förster, F. and Abicht, A. and Schänzer, A. and Schara-Schmidt, U. and Roos, A. and Della Marina, A.
    Human Mutation (2022)
    The synthesis of cytochrome c oxidase 2 (SCO2) gene encodes for a mitochondrial located metallochaperone essential for the synthesis of the cytochrome c oxidase (COX) subunit 2. Recessive mutations in SCO2 have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency and in only four cases with axonal neuropathy. Here, we identified a homozygous pathogenic variant (c.361G > C; p.[Gly121Arg]) in SCO2 in two brothers with isolated axonal motor neuropathy. To address pathogenicity of the amino acid substitution, biochemical studies were performed and revealed increased level of the mutant SCO2-protein and dysregulation of COX subunits in leukocytes and moreover unraveled decrease of proteins involved in the manifestation of neuropathies. Hence, our combined data strengthen the concept of SCO2 being causative for a very rare form of axonal neuropathy, expand its molecular genetic spectrum and provide first biochemical insights into the underlying pathophysiology. © 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.
    view abstract10.1002/humu.24338
  • Identification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro
    Le-Trilling, V.T.K. and Mennerich, D. and Schuler, C. and Sakson, R. and Lill, J.K. and Kasarla, S.S. and Kopczynski, D. and Loroch, S. and Flores-Martinez, Y. and Katschinski, B. and Wohlgemuth, K. and Gunzer, M. and Meyer, F. and Phapale, P. and Dittmer, U. and Sickmann, A. and Trilling, M.
    BMC Biology 20 (2022)
    view abstract10.1186/s12915-022-01468-z
  • Inhibition of Src but not Syk causes weak reversal of GPVI-mediated platelet aggregation measured by light transmission aggregometry
    Cheung, H.Y.F. and Moran, L.A. and Sickmann, A. and Heemskerk, J.W.M. and Garcia, Á. and Watson, S.P.
    Platelets 33 (2022)
    Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton’s tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear. © 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.
    view abstract10.1080/09537104.2022.2069235
  • LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus
    Wu, Z. and Berlemann, L.A. and Bader, V. and Sehr, D.A. and Dawin, E. and Covallero, A. and Meschede, J. and Angersbach, L. and Showkat, C. and Michaelis, J.B. and Münch, C. and Rieger, B. and Namgaladze, D. and Herrera, M.G. and Fiesel, F.C. and Springer, W. and Mendes, M. and Stepien, J. and Barkovits, K. and Marcus, K. and Sickmann, A. and Dittmar, G. and Busch, K.B. and Riedel, D. and Brini, M. and Tatzelt, J. and Cali, T. and Winklhofer, K.F.
    EMBO Journal 41 (2022)
    view abstract10.15252/embj.2022112006
  • Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity
    Hesse, J. and Rosse, M.K. and Steckel, B. and Blank-Landeshammer, B. and Idel, S. and Reinders, Y. and Sickmann, A. and Sträter, N. and Schrader, J.
    Purinergic Signalling 18 (2022)
    CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity. © 2021, The Author(s).
    view abstract10.1007/s11302-021-09832-4
  • Novel insights into PORCN mutations, associated phenotypes and pathophysiological aspects
    Arlt, A. and Kohlschmidt, N. and Hentschel, A. and Bartels, E. and Groß, C. and Töpf, A. and Edem, P. and Szabo, N. and Sickmann, A. and Meyer, N. and Schara-Schmidt, U. and Lau, J. and Lochmüller, H. and Horvath, R. and Oktay, Y. and Roos, A. and Hiz, S.
    Orphanet Journal of Rare Diseases 17 (2022)
    Background: Goltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants. Results: We report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes. Conclusions: Our combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS. © 2022, The Author(s).
    view abstract10.1186/s13023-021-02068-w
  • Proteome-Based Clustering Approaches Reveal Phylogenetic Insights into Amphistegina
    Stuhr, M. and Blank-Landeshammer, B. and Meyer, A. and Baumeister, V. and Rahnenführer, J. and Sickmann, A. and Westphal, H.
    Journal of Earth Science (2022)
    Foraminifera are highly diverse and have a long evolutionary history. As key bioindicators, their phylogenetic schemes are of great importance for paleogeographic applications, but may be hard to recognize correctly. The phylogenetic relationships within the prominent genus Amphistegina are still uncertain. Molecular studies on Amphistegina have so far only focused on genetic diversity within single species and suggested a cryptic diversity that demands for further investigations. Besides molecular sequencing-based approaches, different mass spectrometry-based proteomics approaches are increasingly used to give insights into the relationship between samples and organisms, especially as these do not require reference databases. To better understand the relationship of amphisteginids and test different proteomics-based approaches we applied de novo peptide sequencing and similarity clustering to several populations of Amphistegina lobifera, A. lessonii and A. gibbosa. We also analyzed the dominant photosymbiont community to study their influence on holobiont proteomes. Our analyses indicate that especially de novo peptide sequencing allows to reconstruct the relationship among foraminiferal holobionts, although the detected separation of A. gibbosa from A. lessonii and A. lobifera may be partly influenced by their different photosymbiont types. The resulting dendrograms reflect the separation in two lineages previously suggested and provide a basis for future studies. © 2022, China University of Geosciences (Wuhan) and Springer-Verlag GmbH Germany, Part of Springer Nature.
    view abstract10.1007/s12583-022-1609-1
  • Proteomic and morphological insights and clinical presentation of two young patients with novel mutations of BVES (POPDC1)
    Gangfuß, A. and Hentschel, A. and Heil, L. and Gonzalez, M. and Schönecker, A. and Depienne, C. and Nishimura, A. and Zengeler, D. and Kohlschmidt, N. and Sickmann, A. and Schara-Schmidt, U. and Fürst, D.O. and van der Ven, P.F.M. and Hahn, A. and Roos, A. and Schänzer, A.
    Molecular Genetics and Metabolism 136 (2022)
    Popeye domain containing protein 1 (POPDC1) is a highly conserved transmembrane protein essential for striated muscle function and homeostasis. Pathogenic variants in the gene encoding POPDC1 (BVES, Blood vessel epicardial substance) are causative for limb-girdle muscular dystrophy (LGMDR25), associated with cardiac arrhythmia. We report on four affected children (age 7–19 years) from two consanguineous families with two novel pathogenic variants in BVES c.457C>T(p.Q153X) and c.578T>G (p.I193S). Detailed analyses were performed on muscle biopsies from an affected patient of each family including immunofluorescence, electron microscopy and proteomic profiling. Cardiac abnormalities were present in all patients and serum creatine kinase (CK) values were variably elevated despite lack of overt muscle weakness. Detailed histological analysis of skeletal muscle, however indicated a myopathy with reduced sarcolemmal expression of POPDC1 accompanied by altered sarcolemmal and sarcoplasmatic dysferlin and Xin/XIRP1 abundance. At the electron microscopic level, the muscle fiber membrane was focally disrupted. The proteomic signature showed statistically significant dysregulation of 191 proteins of which 173 were increased and 18 were decreased. Gene ontology-term analysis of affected biological processes revealed - among others - perturbation of muscle fibril assembly, myofilament sliding, and contraction as well as transition between fast and slow fibers. In conclusion, these findings demonstrate that the phenotype of LGMDR25 is highly variable and also includes younger children with conduction abnormalities, no apparent muscular problems, and only mildly elevated CK values. Biochemical studies suggest that BVES mutations causing loss of functional POPDC1 can impede striated muscle function by several mechanisms. © 2022 Elsevier Inc.
    view abstract10.1016/j.ymgme.2022.05.005
  • Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear
    Huang, J. and Jooss, N.J. and Fernández, D.I. and Sickmann, A. and García, Á. and Wichapong, K. and Dijkgraaf, I. and Heemskerk, J.W.M.
    International Journal of Molecular Sciences 23 (2022)
    Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling. © 2022 by the authors.
    view abstract10.3390/ijms23158688
  • Skeletal muscle provides the immunological micro-milieu for specific plasma cells in anti-synthetase syndrome-associated myositis
    Preuße, C. and Paesler, B. and Nelke, C. and Cengiz, D. and Müntefering, T. and Roos, A. and Amelin, D. and Allenbach, Y. and Uruha, A. and Dittmayer, C. and Hentschel, A. and Pawlitzki, M. and Hoffmann, S. and Timm, S. and Louis, S.L. and Dengler, N.F. and Wiendl, H. and Lünemann, J.D. and Sickmann, A. and Hervier, B. and Meuth, S.G. and Schneider, U. and Schänzer, A. and Krause, S. and Tomaras, S. and Feist, E. and Hasseli, R. and Goebel, H.-H. and Gallay, L. and Streichenberger, N. and Benveniste, O. and Stenzel, W. and Ruck, T.
    Acta Neuropathologica 144 (2022)
    Anti-synthetase syndrome (ASyS)-associated myositis is a major subgroup of the idiopathic inflammatory myopathies (IIM) and is characterized by disease chronicity with musculoskeletal, dermatological and pulmonary manifestations. One of eight autoantibodies against the aminoacyl-transferase RNA synthetases (ARS) is detectable in the serum of affected patients. However, disease-specific therapeutic approaches have not yet been established. To obtain a deeper understanding of the underlying pathogenesis and to identify putative therapeutic targets, we comparatively investigated the most common forms of ASyS associated with anti-PL-7, anti-PL-12 and anti-Jo-1. Our cohort consisted of 80 ASyS patients as well as healthy controls (n = 40), diseased controls (n = 40) and non-diseased controls (n = 20). We detected a reduced extent of necrosis and regeneration in muscle biopsies from PL-12+ patients compared to Jo-1+ patients, while PL-7+ patients had higher capillary dropout in biopsies of skeletal muscle. Aside from these subtle alterations, no significant differences between ASyS subgroups were observed. Interestingly, a tissue-specific subpopulation of CD138+ plasma cells and CXCL12+/CXCL13+CD20+ B cells common to ASyS myositis were identified. These cells were localized in the endomysium associated with alkaline phosphatase+ activated mesenchymal fibroblasts and CD68+MHC-II+CD169+ macrophages. An MHC-I+ and MHC-II+ MxA negative type II interferon-driven milieu of myofiber activation, topographically restricted to the perifascicular area and the adjacent perimysium, as well as perimysial clusters of T follicular helper cells defined an extra-medullary immunological niche for plasma cells and activated B cells. Consistent with this, proteomic analyses of muscle tissues from ASyS patients demonstrated alterations in antigen processing and presentation. In-depth immunological analyses of peripheral blood supported a B-cell/plasma-cell-driven pathology with a shift towards immature B cells, an increase of B-cell-related cytokines and chemokines, and activation of the complement system. We hypothesize that a B-cell-driven pathology with the presence and persistence of a specific subtype of plasma cells in the skeletal muscle is crucially involved in the self-perpetuating chronicity of ASyS myositis. This work provides the conceptual framework for the application of plasma-cell-targeting therapies in ASyS myositis. © 2022, The Author(s).
    view abstract10.1007/s00401-022-02438-z
  • Targeting early stages of cardiotoxicity from anti-PD1 immune checkpoint inhibitor therapy
    Michel, L. and Helfrich, I. and Hendgen-Cotta, U.B. and Mincu, R.-I. and Korste, S. and Mrotzek, S.M. and Spomer, A. and Odersky, A. and Rischpler, C. and Herrmann, K. and Umutlu, L. and Coman, C. and Ahrends, R. and Sickmann, A. and Löffek, S. and Livingstone, E. and Ugurel, S. and Zimmer, L. and Gunzer, M. and Schadendorf, D. and Totzeck, M. and Rassaf, T.
    European heart journal 43 (2022)
    AIMS: Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease. METHODS AND RESULTS: We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy. CONCLUSIONS: Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email:
    view abstract10.1093/eurheartj/ehab430
  • The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression
    Bujanic, L. and Shevchuk, O. and von Kügelgen, N. and Kalinina, A. and Ludwik, K. and Koppstein, D. and Zerna, N. and Sickmann, A. and Chekulaeva, M.
    RNA 28 (2022)
    SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem–loop (SL1) in the viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19, and 20) and another within NSP1 (R124), which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing. © 2022 Bujanic et al.
    view abstract10.1261/RNA.079086.121
  • The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro
    Merches, K. and Breunig, L. and Fender, J. and Brand, T. and Bätz, V. and Idel, S. and Kollipara, L. and Reinders, Y. and Sickmann, A. and Mally, A. and Lorenz, K.
    Archives of Toxicology 96 (2022)
    Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coronavirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6–3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24–48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease. © 2022, The Author(s).
    view abstract10.1007/s00204-022-03306-1
  • Toward Zero Variance in Proteomics Sample Preparation: Positive-Pressure FASP in 96-Well Format (PF96) Enables Highly Reproducible, Time- and Cost-Efficient Analysis of Sample Cohorts
    Loroch, S. and Kopczynski, D. and Schneider, A.C. and Schumbrutzki, C. and Feldmann, I. and Panagiotidis, E. and Reinders, Y. and Sakson, R. and Solari, F.A. and Vening, A. and Swieringa, F. and Heemskerk, J.W.M. and Grandoch, M. and Dandekar, T. and Sickmann, A.
    Journal of Proteome Research 21 (2022)
    As novel liquid chromatography-mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36-60 μg result in optimal peptide recovery, but lower amounts ≥3 μg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research. © 2022 American Chemical Society. All rights reserved.
    view abstract10.1021/acs.jproteome.1c00706
  • A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions
    Madhavan, B.K. and Han, Z. and Sickmann, A. and Pepperkok, R. and Nawroth, P.P. and Kumar, V.
    STAR Protocols 2 (2021)
    With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2017, 2020). © 2021 The Author(s)
    view abstract10.1016/j.xpro.2021.100700
  • ANXA7 Regulates Platelet Lipid Metabolism and Ca2+Release in Arterial Thrombosis
    Manke, M.-C. and Geue, S. and Coman, C. and Peng, B. and Kollotzek, F. and Munzer, P. and Walker, B. and Huber, S.M. and Rath, D. and Sickmann, A. and Stegner, D. and Duerschmied, D. and Lang, F. and Nieswandt, B. and Gawaz, M. and Ahrends, R. and Borst, O.
    Circulation Research (2021)
    Rationale: Platelet activation after contact to subendothelial collagen leads to acute arterial thrombosis. ANXA7 (Annexin A7) is a phospholipid-binding protein participating in the regulation of intracellular Ca2+and exocytosis. Objective: The present study aimed to determine the role of ANXA7 in platelet Ca2+signaling and lipid metabolism during platelet activation in arterial thrombosis using the ANXA7 inhibitor 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) and gene-targeted mice lacking Anxa7 (Anxa7-/-). Methods and Results: ANXA7 is strongly expressed in platelets. Functionally, luminescence aggregometry revealed significantly abrogated aggregation and secretion of ABO-treated or Anxa7-/-platelets when compared with untreated or Anxa7+/+platelets after activation with collagen or the GPVI (glycoprotein VI)-specific agonist collagen-related peptide. Furthermore, while both thrombus formation on collagen-coated surfaces under high arterial shear rates in ABO-treated or Anxa7-deficient whole blood, and thrombotic vascular occlusion after FeCl3-induced injury in vivo in Anxa7-/-bone marrow chimeric mice were significantly diminished, no prolongation of bleeding time was observed in ABO-treated or Anxa7-/-mice. Fura-2-AM spectrofluorimetry unraveled a blunted [Ca2+]iincrease in ABO-treated or Anxa7-/-platelets after GPVI stimulation. Due to an abolished phospholipase Cγ2 phosphorylation, Anxa7-/-platelets displayed abrogated intracellular Ca2+mobilization following collagen-related peptide-dependent platelet activation. Quantitative lipidomics analysis further revealed that ANXA7 critically affects platelet oxylipin metabolism following GPVI-dependent platelet activation. Anxa7-/-platelets showed a significantly reduced generation of several bioactive metabolites, particularly thromboxane A2and 12(S)-hydroxy-eicosatetraenoic acid. Finally, defective phospholipase Cγ2 phosphorylation and blunted [Ca2+]iincrease in Anxa7-/-platelets could be rescued by exogenous addition of 12(S)-hydroxy-eicosatetraenoic acid, indicating that ANXA7 is a critical regulator of the platelet 12-lipoxygenase in GPVI-dependent platelet Ca2+signaling during arterial thrombosis. Conclusions: The present study unravels ANXA7 as a regulator of oxylipin metabolism and Ca2+-dependent platelet activation downstream of GPVI. ANXA7 plays an important role in platelet signaling during arterial thrombosis and thus may reflect a promising target for novel antiplatelet strategies. © 2021 Lippincott Williams and Wilkins. All rights reserved.
    view abstract10.1161/CIRCRESAHA.121.319207
  • Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions
    Huang, J. and Swieringa, F. and Solari, F.A. and Provenzale, I. and Grassi, L. and De Simone, I. and Baaten, C.C.F.M.J. and Cavill, R. and Sickmann, A. and Frontini, M. and Heemskerk, J.W.M.
    Scientific Reports 11 (2021)
    Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43–0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease. © 2021, The Author(s).
    view abstract10.1038/s41598-021-91661-x
  • eIF5A hypusination, boosted by dietary spermidine, protects from premature brain aging and mitochondrial dysfunction
    Liang, Y. and Piao, C. and Beuschel, C.B. and Toppe, D. and Kollipara, L. and Bogdanow, B. and Maglione, M. and Lützkendorf, J. and See, J.C.K. and Huang, S. and Conrad, T.O.F. and Kintscher, U. and Madeo, F. and Liu, F. and Sickmann, A. and Sigrist, S.J.
    Cell Reports 35 (2021)
    Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). Here, we show that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline. © 2021 The Author(s)
    view abstract10.1016/j.celrep.2021.108941
  • Exercise prevents fatty liver by modifying the compensatory response of mitochondrial metabolism to excess substrate availability
    Hoene, M. and Kappler, L. and Kollipara, L. and Hu, C. and Irmler, M. and Bleher, D. and Hoffmann, C. and Beckers, J. and Hrabě de Angelis, M. and Häring, H.-U. and Birkenfeld, A.L. and Peter, A. and Sickmann, A. and Xu, G. and Lehmann, R. and Weigert, C.
    Molecular Metabolism 54 (2021)
    Objective: Liver mitochondria adapt to high-calorie intake. We investigated how exercise alters the early compensatory response of mitochondria, thus preventing fatty liver disease as a long-term consequence of overnutrition. Methods: We compared the effects of a steatogenic high-energy diet (HED) for six weeks on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome, and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high-resolution respirometry. Results: HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism, and lipogenesis. Training also counteracted the HED-induced glucose intolerance, the increase in fasting insulin, and in liver fat by lowering diacylglycerol species and c-Jun N-terminal kinase (JNK) phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content. Conclusion: We provide a comprehensive insight into the early adaptations of mitochondria in the liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity. © 2021 The Author(s)
    view abstract10.1016/j.molmet.2021.101359
  • Exome reanalysis and proteomic profiling identified TRIP4 as a novel cause of cerebellar hypoplasia and spinal muscular atrophy (PCH1)
    Töpf, A. and Pyle, A. and Griffin, H. and Matalonga, L. and Schon, K. and Sickmann, A. and Schara-Schmidt, U. and Hentschel, A. and Chinnery, P.F. and Kölbel, H. and Roos, A. and Horvath, R. and Solve-RD SNV-indel working group and Solve-RD DITF-euroNMD
    European journal of human genetics : EJHG 29 (2021)
    TRIP4 is one of the subunits of the transcriptional coregulator ASC-1, a ribonucleoprotein complex that participates in transcriptional coactivation and RNA processing events. Recessive variants in the TRIP4 gene have been associated with spinal muscular atrophy with bone fractures as well as a severe form of congenital muscular dystrophy. Here we present the diagnostic journey of a patient with cerebellar hypoplasia and spinal muscular atrophy (PCH1) and congenital bone fractures. Initial exome sequencing analysis revealed no candidate variants. Reanalysis of the exome data by inclusion in the Solve-RD project resulted in the identification of a homozygous stop-gain variant in the TRIP4 gene, previously reported as disease-causing. This highlights the importance of analysis reiteration and improved and updated bioinformatic pipelines. Proteomic profile of the patient's fibroblasts showed altered RNA-processing and impaired exosome activity supporting the pathogenicity of the detected variant. In addition, we identified a novel genetic form of PCH1, further strengthening the link of this characteristic phenotype with altered RNA metabolism. © 2021. The Author(s).
    view abstract10.1038/s41431-021-00851-8
  • Future perspectives on in-vitro diagnosis of drug allergy by the lymphocyte transformation test
    Fatangare, A. and Glässner, A. and Sachs, B. and Sickmann, A.
    Journal of Immunological Methods 495 (2021)
    This article aims to envisage future perspectives of the lymphocyte transformation test (LTT). We describe the select innovative techniques, which can be integrated at different stages of the LTT to potentially improve the sensitivity, specificity, or practicability of the LTT. We first focus upon the cell sorting techniques comprising immunomagnetic cell separation and flow cytometry, which can be implemented prior and after the LTT culturing step to concentrate and quantify specific immune cell types. Further, we elaborate upon three important omics techniques such as transcriptomics, proteomics, and metabolomics, which can be integrated downstream of the LTT to analyze molecular changes in specific immune cells following drug induced activation and proliferation. We also develop visions, how state of the art techniques used in other scientific fields, can be transferred and applied in the context of in-vitro detection of drug allergy. © 2021
    view abstract10.1016/j.jim.2021.113072
  • Generation of a humanized FXII knock-in mouse—A powerful model system to test novel anti-thrombotic agents
    Beck, S. and Stegner, D. and Loroch, S. and Baig, A.A. and Göb, V. and Schumbrutzki, C. and Eilers, E. and Sickmann, A. and May, F. and Nolte, M.W. and Panousis, C. and Nieswandt, B.
    Journal of Thrombosis and Haemostasis 19 (2021)
    Background: Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. Objective: The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. Methods: A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. Results: These hF12KI mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12KI mice in an arterial thrombosis model without affecting bleeding times. Conclusion: These data establish the newly generated hF12KI mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors. © 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.
    view abstract10.1111/jth.15488
  • Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery
    Walter, C. and Marada, A. and Suhm, T. and Ernsberger, R. and Muders, V. and Kücükköse, C. and Sánchez-Martín, P. and Hu, Z. and Aich, A. and Loroch, S. and Solari, F.A. and Poveda-Huertes, D. and Schwierzok, A. and Pommerening, H. and Matic, S. and Brix, J. and Sickmann, A. and Kraft, C. and Dengjel, J. and Dennerlein, S. and Brummer, T. and Vögtle, F.-N. and Meisinger, C.
    Nature Communications 12 (2021)
    The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome. © 2021, The Author(s).
    view abstract10.1038/s41467-021-24426-9
  • Homozygous WASHC4 variant in two sisters causes a syndromic phenotype defined by dysmorphisms, intellectual disability, profound developmental disorder, and skeletal muscle involvement
    Gangfuß, A. and Czech, A. and Hentschel, A. and Münchberg, U. and Horvath, R. and Töpf, A. and O'Heir, E. and Lochmüller, H. and Stehling, F. and Kiewert, C. and Sickmann, A. and Kuechler, A. and Kaiser, F.J. and Kölbel, H. and Christiansen, J. and Schara-Schmidt, U. and Roos, A.
    Journal of Pathology (2021)
    Recessive variants in WASHC4 are linked to intellectual disability complicated by poor language skills, short stature, and dysmorphic features. The protein encoded by WASHC4 is part of the Wiskott–Aldrich syndrome protein and SCAR homolog family, co-localizes with actin in cells, and promotes Arp2/3-dependent actin polymerization in vitro. Functional studies in a zebrafish model suggested that WASHC4 knockdown may also affect skeletal muscles by perturbing protein clearance. However, skeletal muscle involvement has not been reported so far in patients, and precise biochemical studies allowing a deeper understanding of the molecular etiology of the disease are still lacking. Here, we report two siblings with a homozygous WASHC4 variant expanding the clinical spectrum of the disease and provide a phenotypical comparison with cases reported in the literature. Proteomic profiling of fibroblasts of the WASHC4-deficient patient revealed dysregulation of proteins relevant for the maintenance of the neuromuscular axis. Immunostaining on a muscle biopsy derived from the same patient confirmed dysregulation of proteins relevant for proper muscle function, thus highlighting an affliction of muscle cells upon loss of functional WASHC4. The results of histological and coherent anti-Stokes Raman scattering microscopic studies support the concept of a functional role of the WASHC4 protein in humans by altering protein processing and clearance. The proteomic analysis confirmed key molecular players in vitro and highlighted, for the first time, the involvement of skeletal muscle in patients. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
    view abstract10.1002/path.5812
  • Identification of the factor XII contact activation site enables sensitive coagulation diagnostics
    Heestermans, M. and Naudin, C. and Mailer, R.K. and Konrath, S. and Klaetschke, K. and Jämsä, A. and Frye, M. and Deppermann, C. and Pula, G. and Kuta, P. and Friese, M.A. and Gelderblom, M. and Sickmann, A. and Preston, R.J.S. and Nofer, J.-R. and Rose-John, S. and Butler, L.M. and Salomon, O. and Stavrou, E.X. and Renné, T.
    Nature Communications 12 (2021)
    Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI. © 2021, The Author(s).
    view abstract10.1038/s41467-021-25888-7
  • Lymphocyte transformation test: History and current approaches
    Sachs, B. and Fatangare, A. and Sickmann, A. and Glässner, A.
    Journal of Immunological Methods 493 (2021)
    Drug-induced hypersensitivity reactions encompass a variety of different clinical phenotypes ranging from harmless rashes to fatal reactions. They can be classified into allergic (i.e. drug allergy) and non-allergic reactions (i.e. non-allergic hypersensitivity). Drug allergies in turn can either be antibody (e.g. IgE) or T cell-mediated. One of the diagnostic tools for the in vitro detection of drug allergy is the lymphocyte transformation test (LTT) which is based on the activation and expansion of the drug-specific memory T cells following co-incubation of the patient's peripheral mononuclear cells (PMBC) with the suspected drug in vitro. The read-out parameter in the classical LTT is T cell proliferation which can be measured as counts per minute following the addition of radiolabeled thymidine to the cell culture. However, in the course of time different modifications of the classical LTT with regard to the read-out parameters and methods have been proposed. Likewise, variations of the LTT platform itself have been described in the literature. This review article describes the development of the classical LTT and its use in the context of drug allergy detection and summarizes the modifications which have been published over time. © 2021 The Authors
    view abstract10.1016/j.jim.2021.113036
  • MARCKS affects cell motility and response to BTK inhibitors in CLL
    Beckmann, L. and Berg, V. and Dickhut, C. and Sun, C. and Merkel, O. and Bloehdorn, J. and Robrecht, S. and Seifert, M. and da Palma Guerreiro, A. and Claasen, J. and Loroch, S. and Oliverio, M. and Underbayev, C. and Vaughn, L. and Thomalla, D. and Hülsemann, M.F. and Tausch, E. and Fischer, K. and Fink, A.M. and Eichhorst, B. and Sickmann, A. and Wendtner, C.M. and Stilgenbauer, S. and Hallek, M. and Wiestner, A. and Zahedi, R.P. and Frenzel, L.P.
    Blood 138 (2021)
    Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib. © 2021 American Society of Hematology
    view abstract10.1182/blood.2020009165
  • Molecular proteomics and signalling of human platelets in health and disease
    Huang, J. and Zhang, P. and Solari, F.A. and Sickmann, A. and Garcia, A. and Jurk, K. and Heemskerk, J.W.M.
    International Journal of Molecular Sciences 22 (2021)
    Platelets are small anucleate blood cells that play vital roles in haemostasis and thrombosis, besides other physiological and pathophysiological processes. These roles are tightly regulated by a complex network of signalling pathways. Mass spectrometry-based proteomic techniques are contributing not only to the identification and quantification of new platelet proteins, but also reveal post-translational modifications of these molecules, such as acetylation, glycosylation and phosphorylation. Moreover, target proteomic analysis of platelets can provide molecular biomarkers for genetic aberrations with established or non-established links to platelet dysfunctions. In this report, we review 67 reports regarding platelet proteomic analysis and signalling on a molecular base. Collectively, these provide detailed insight into the: (i) technical developments and limitations of the assessment of platelet (sub)proteomes; (ii) molecular protein changes upon ageing of platelets; (iii) complexity of platelet signalling pathways and functions in response to collagen, rhodocytin, thrombin, thromboxane A2 and ADP; (iv) proteomic effects of endothelial-derived mediators such as prostacyclin and the anti-platelet drug aspirin; and (v) molecular protein changes in platelets from patients with congenital disorders or cardiovascular disease. However, sample sizes are still low and the roles of differentially expressed proteins are often unknown. Based on the practical and technical possibilities and limitations, we provide a perspective for further improvements of the platelet proteomic field. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ijms22189860
  • Mouse Quantitative Proteomics Knowledgebase: Reference protein concentration ranges in 20 mouse tissues using 5000 quantitative proteomics assays
    Mohammed, Y. and Bhowmick, P. and Michaud, S.A. and Sickmann, A. and Borchers, C.H.
    Bioinformatics 37 (2021)
     : MotivationLaboratory mouse is the most used animal model in biological research, largely due to its high conserved synteny with human. Researchers use mice to answer various questions ranging from determining a pathological effect of knocked out/in gene to understanding drug metabolism. Our group developed >5000 quantitative targeted proteomics assays for 20 mouse tissues and determined the concentration ranges of a total of >1600 proteins using heavy labeled internal standards. We describe here MouseQuaPro; a knowledgebase that hosts this collection of carefully curated experimental data. ResultsThe web-based application includes protein concentrations from >700 mouse tissue samples from three common research strains, corresponding to >200k experimentally determined concentrations. The knowledgebase integrates the assay and protein concentration information with their human orthologs, functional and molecular annotations, biological pathways, related human diseases and known gene expressions. At its core are the protein concentration ranges, which provide insights into (dis)similarities between tissues, strains and sexes. MouseQuaPro implements advanced search as well as filtering functionalities with a simple interface and interactive visualization. This information-rich resource provides an initial map of protein absolute concentration in mouse tissues and allows guided design of proteomics phenotyping experiments. The knowledgebase is available on © 2021 The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:
    view abstract10.1093/bioinformatics/btab018
  • Muscular and molecular pathology associated with SPATA5 deficiency in a child with EHLMRS
    Braun, F. and Hentschel, A. and Sickmann, A. and Marteau, T. and Hertel, S. and Förster, F. and Prokisch, H. and Wagner, M. and Wortmann, S. and Della Marina, A. and Kölbel, H. and Roos, A. and Schara‐schmidt, U.
    International Journal of Molecular Sciences 22 (2021)
    Mutations in the SPATA5 gene are associated with epilepsy, hearing loss and mental retardation syndrome (EHLMRS). While SPATA5 is ubiquitously expressed and is attributed a role within mitochondrial morphogenesis during spermatogenesis, there is only limited knowledge about the associated muscular and molecular pathology. This study reports on a comprehensive workup of muscular pathology, including proteomic profiling and microscopic studies, performed on an 8‐year‐old girl with typical clinical presentation of EHLMRS, where exome analysis revealed two clinically relevant, compound‐heterozygous variants in SPATA5. Proteomic profiling of a quadriceps biopsy showed the dysregulation of 82 proteins, out of which 15 were localized in the mitochondrion, while 19 were associated with diseases presenting with phenotypical overlap to EHLMRS. Histological staining of our patient’s muscle biopsy hints towards mitochondrial pathology, while the identification of dysregulated proteins attested to the vulnerability of the cell beyond the mitochondria. Through our study we provide insights into the molecular etiology of EHLMRS and provide further evidence for a muscle pathology associated with SPATA5 deficiency, including a pathological histochemical pattern accompanied by dysregulated protein expression. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ijms22157835
  • Phenotypical and myopathological consequences of compound heterozygous missense and nonsense variants in slc18a3
    Marina, A.D. and Arlt, A. and Schara-Schmidt, U. and Depienne, C. and Gangfuß, A. and Kölbel, H. and Sickmann, A. and Freier, E. and Kohlschmidt, N. and Hentschel, A. and Weis, J. and Czech, A. and Grüneboom, A. and Roos, A.
    Cells 10 (2021)
    Background: Presynaptic forms of congenital myasthenic syndromes (CMS) due to pathogenic variants in SLC18A3 impairing the synthesis and recycling of acetylcholine (ACh) have recently been described. SLC18A3 encodes the vesicular ACh transporter (VAChT), modulating the active transport of ACh at the neuromuscular junction, and homozygous loss of VAChT leads to lethality. Methods: Exome sequencing (ES) was carried out to identify the molecular genetic cause of the disease in a 5-year-old male patient and histological, immunofluorescence as well as electron-and CARS-microscopic studies were performed to delineate the muscle pathology, which has so far only been studied in VAChT-deficient animal models. Results: ES unraveled compound heterozygous missense and nonsense variants (c.315G>A, p.Trp105* and c.1192G>C, p.Asp398His) in SLC18A3. Comparison with already-published cases suggests a more severe phenotype including impaired motor and cognitive development, possibly related to a more severe effect of the nonsense variant. Therapy with pyridostigmine was only partially effective while 3,4 diaminopyridine showed no effect. Microscopic investigation of the muscle biopsy revealed reduced fibre size and a significant accumulation of lipid droplets. Conclusions: We suggest that nonsense variants have a more detrimental impact on the clinical manifestation of SLC18A3-associated CMS. The impact of pathogenic SLC18A3 variants on muscle fibre integrity beyond the effect of denervation is suggested by the build-up of lipid aggregates. This in turn implicates the importance of proper VAChT-mediated synthesis and recycling of ACh for lipid homeostasis in muscle cells. This hypothesis is further supported by the pathological observations obtained in previously published VAChT-animal models. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/cells10123481
  • Protein signature of human skin fibroblasts allows the study of the molecular etiology of rare neurological diseases
    Hentschel, A. and Czech, A. and Münchberg, U. and Freier, E. and Schara-Schmidt, U. and Sickmann, A. and Reimann, J. and Roos, A.
    Orphanet Journal of Rare Diseases 16 (2021)
    Background: The elucidation of pathomechanisms leading to the manifestation of rare (genetically caused) neurological diseases including neuromuscular diseases (NMD) represents an important step toward the understanding of the genesis of the respective disease and might help to define starting points for (new) therapeutic intervention concepts. However, these “discovery studies” are often limited by the availability of human biomaterial. Moreover, given that results of next-generation-sequencing approaches frequently result in the identification of ambiguous variants, testing of their pathogenicity is crucial but also depending on patient-derived material. Methods: Human skin fibroblasts were used to generate a spectral library using pH8-fractionation of followed by nano LC-MS/MS. Afterwards, Allgrove-patient derived fibroblasts were subjected to a data independent acquisition approach. In addition, proteomic signature of an enriched nuclear protein fraction was studied. Proteomic findings were confirmed by immunofluorescence in a muscle biopsy derived from the same patient and cellular lipid homeostasis in the cause of Allgrove syndrome was analysed by fluorescence (BODIPY-staining) and coherent anti-Stokes Raman scattering (CARS) microscopy. Results: To systematically address the question if human skin fibroblasts might serve as valuable biomaterial for (molecular) studies of NMD, we generated a protein library cataloguing 8280 proteins including a variety of such linked to genetic forms of motoneuron diseases, congenital myasthenic syndromes, neuropathies and muscle disorders. In silico-based pathway analyses revealed expression of a diversity of proteins involved in muscle contraction and such decisive for neuronal function and maintenance suggesting the suitability of human skin fibroblasts to study the etiology of NMD. Based on these findings, next we aimed to further demonstrate the suitability of this in vitro model to study NMD by a use case: the proteomic signature of fibroblasts derived from an Allgrove-patient was studied. Dysregulation of paradigmatic proteins could be confirmed in muscle biopsy of the patient and protein-functions could be linked to neurological symptoms known for this disease. Moreover, proteomic investigation of nuclear protein composition allowed the identification of protein-dysregulations according with structural perturbations observed in the muscle biopsy. BODIPY-staining on fibroblasts and CARS microscopy on muscle biopsy suggest altered lipid storage as part of the underlying disease etiology. Conclusions: Our combined data reveal that human fibroblasts may serve as an in vitro system to study the molecular etiology of rare neurological diseases exemplified on Allgrove syndrome in an unbiased fashion. © 2021, The Author(s).
    view abstract10.1186/s13023-020-01669-1
  • Proteomics: A Tool to Study Platelet Function
    Shevchuk, O. and Begonja, A.J. and Gambaryan, S. and Totzeck, M. and Rassaf, T. and Huber, T.B. and Greinacher, A. and Renne, T. and Sickmann, A.
    International journal of molecular sciences 22 (2021)
    Platelets are components of the blood that are highly reactive, and they quickly respond to multiple physiological and pathophysiological processes. In the last decade, it became clear that platelets are the key components of circulation, linking hemostasis, innate, and acquired immunity. Protein composition, localization, and activity are crucial for platelet function and regulation. The current state of mass spectrometry-based proteomics has tremendous potential to identify and quantify thousands of proteins from a minimal amount of material, unravel multiple post-translational modifications, and monitor platelet activity during drug treatments. This review focuses on the role of proteomics in understanding the molecular basics of the classical and newly emerging functions of platelets. including the recently described role of platelets in immunology and the development of COVID-19.The state-of-the-art proteomic technologies and their application in studying platelet biogenesis, signaling, and storage are described, and the potential of newly appeared trapped ion mobility spectrometry (TIMS) is highlighted. Additionally, implementing proteomic methods in platelet transfusion medicine, and as a diagnostic and prognostic tool, is discussed.
    view abstract10.3390/ijms22094776
  • Quantitative Proteome Data Analysis of Tandem Mass Tags Labeled Samples
    Pagel, O. and Kollipara, L. and Sickmann, A.
    Methods in Molecular Biology 2228 (2021)
    In mass spectrometry-based proteomics, relative quantitative approaches enable differential protein abundance analysis. Isobaric labeling strategies, such as tandem mass tags (TMT), provide simultaneous quantification of several samples (e.g., up to 16 using 16plex TMTpro) owing to its multiplexing capability. This technology improves sample throughput and thereby minimizes both measurement time and overall experimental variation. However, TMT-based MS data processing and statistical analysis are probably the crucial parts of this pipeline to obtain reliable, plausible, and significantly quantified results. Here, we provide a step-by-step guide to the analysis and evaluation of TMT quantitative proteomics data. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
    view abstract10.1007/978-1-0716-1024-4_28
  • Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor
    Straka, T. and Schröder, C. and Roos, A. and Kollipara, L. and Sickmann, A. and Williams, M.P.I. and Hafner, M. and Khan, M.M. and Rudolf, R.
    Frontiers in Physiology 12 (2021)
    Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction. © Copyright © 2021 Straka, Schröder, Roos, Kollipara, Sickmann, Williams, Hafner, Khan and Rudolf.
    view abstract10.3389/fphys.2021.626707
  • Tandem Mass Tags for Comparative and Discovery Proteomics
    Pagel, O. and Kollipara, L. and Sickmann, A.
    Methods in Molecular Biology 2228 (2021)
    Relative or comparative proteomics provides valuable insights about the altered protein abundances across different biological samples in a single (labeled) or series (label-free) of LC–MS measurement(s). Chemical labeling of peptides using isobaric mass tags for identification and quantification of different proteomes simultaneously has become a routine in the so-called discovery proteomics in the past decade. One of the earliest isobaric tags-based technologies is TMT (tandem mass tags), which relies on the comparison of the unique “reporter ions” intensities for relative peptide/protein quantification. This differential labeling approach has evolved over time with respect to its multiplexing capability, i.e., from just 2 samples (TMTduplex) to 10 samples (TMT10plex) and a nowadays of up to 16 samples (TMTpro 16plex). Here, we describe a straightforward protocol to perform relatively deep proteome quantitative analyses using TMT10plex. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
    view abstract10.1007/978-1-0716-1024-4_9
  • Targeted Phosphoinositides Analysis Using High-Performance Ion Chromatography-Coupled Selected Reaction Monitoring Mass Spectrometry
    Cheung, H.Y.F. and Coman, C. and Westhoff, P. and Manke, M. and Sickmann, A. and Borst, O. and Gawaz, M. and Watson, S.P. and Heemskerk, J.W.M. and Ahrends, R.
    Journal of Proteome Research 20 (2021)
    Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography-mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides. © 2021 American Chemical Society.
    view abstract10.1021/acs.jproteome.1c00017
  • Targeted quantification of phosphorylation sites identifies stripak-dependent phosphorylation of the hippo pathway-related kinase smkin3
    Stein, V. and Blank-Landeshammer, B. and Märker, R. and Sickmann, A. and Kück, U.
    mBio 12 (2021)
    We showed recently that the germinal center kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septa-tion initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ∆Smkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ∆Smkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on con-trolled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation. IMPORTANCE Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological ac-tivity. Involved in this modification process is the recently identified striatin-interact-ing phosphatase and kinase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in signaling components that are linked to the STRIPAK complex. Using the filamentous fungus Sordaria macrospora, we provide evidence for the phosphorylation-dependent role of the Hippo-like germinal center kinase SmKIN3, which controls septum formation, and localize it in a time-dependent manner on septa at the hyphal tip. © 2021 Stein et al.
    view abstract10.1128/mBio.00658-21
  • Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins
    Pech, S. and Rehberg, M. and Janke, R. and Benndorf, D. and Genzel, Y. and Muth, T. and Sickmann, A. and Rapp, E. and Reichl, U.
    Applied Microbiology and Biotechnology (2021)
    Abstract: Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. Key points: • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells © 2021, The Author(s).
    view abstract10.1007/s00253-021-11150-z
  • Xenotropic and polytropic retrovirus receptor 1 regulates procoagulant platelet polyphosphate
    Mailer, R.K. and Allende, M. and Heestermans, M. and Schweizer, M. and Deppermann, C. and Frye, M. and Pula, G. and Odeberg, J. and Gelderblom, M. and Rose-John, S. and Sickmann, A. and Blankenberg, S. and Huber, T.B. and Kubisch, C. and Maas, C. and Gambaryan, S. and Firsov, D. and Stavrou, E.X. and Butler, L.M. and Renné, T.
    Blood 137 (2021)
    Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis. Key Points: • Xenotropic and polytropic retrovirus receptor 1 (XPR1) is a major phosphate exporter in platelets. • Inhibiting XPR1 in platelets increases procoagulant polyphosphate levels and augments arterial and venous thrombosis in mice. © 2021 American Society of Hematology
    view abstract10.1182/blood.2019004617
  • BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation
    Volz, J. and Kusch, C. and Beck, S. and Popp, M. and Vögtle, T. and Meub, M. and Scheller, I. and Heil, H.S. and Preu, J. and Schuhmann, M.K. and Hemmen, K. and Premsler, T. and Sickmann, A. and Heinze, K.G. and Stegner, D. and Stoll, G. and Braun, A. and Sauer, M. and Nieswandt, B.
    Journal of Clinical Investigation 130 (2020)
    Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings. Copyright: © 2020, American Society for Clinical Investigation.
    view abstract10.1172/JCI136457
  • Coactosin-like 1 integrates signaling critical for shear-dependent thrombus formation in mouse platelets
    Scheller, I. and Stritt, S. and Beck, S. and Peng, B. and Pleines, I. and Heinze, K.G. and Braun, A. and Otto, O. and Ahrends, R. and Sickmann, A. and Bender, M. and Nieswandt, B.
    Haematologica 105 (2020)
    Platelet aggregate formation is a multistep process involving receptormediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1-/-) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1-/- platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1-/- platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis. © 2020 Ferrata Storti Foundation.
    view abstract10.3324/haematol.2019.225516
  • Cutting the Gordian knot: early and complete amino acid sequence confirmation of class II lasso peptides by HCD fragmentation
    Jarmusch, S.A. and Feldmann, I. and Blank-Landeshammer, B. and Cortés-Albayay, C. and Castro, J.F. and Andrews, B. and Asenjo, J.A. and Sickmann, A. and Ebel, R. and Jaspars, M.
    Journal of Antibiotics 73 (2020)
    Lasso peptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Their proteolytic and thermal stability alongside their growing potential as therapeutics has increased attention to these antimicrobial peptides. With the advent of genome mining, the discovery of RiPPs allows for the accurate prediction of putatively encoded structures, however, MSn experiments only provide partial sequence confirmation, therefore 2D NMR experiments are necessary for characterisation. Multiple MS/MS techniques were applied to two structurally characterized lasso peptides, huascopeptin and leepeptin, and one uncharacterized lasso peptide, citrulassin C, which was not isolable in sufficient quantity for NMR analysis. We have shown that MS2 can be used to elucidate the full amino acid sequences previously predicted with genome mining for this compound class. HCD was able to open the macrocycles and fragment the newly opened linear peptides, confirming the complete amino acid sequences of the characterised lasso peptides. In addition, to determine if this technique could be applied at the earliest stages of the isolation process, we targeted a lasso peptide found by genome mining, citrulassin C, and were able to fully elucidate the amino acid sequence using only MS2 from a semi-crude extract of Streptomyces huasconensis HST28T. © 2020, The Author(s), under exclusive licence to the Japan Antibiotics Research Association.
    view abstract10.1038/s41429-020-00369-z
  • Data Independent Acquisition Mass Spectrometry for Proteomic Advances into Isolated Methylmalonic Acidemia
    Costanzo, M. and Caterino, M. and Cevenini, A. and Kollipara, L. and Shevchuk, O. and Nguyen, C.D.L. and Sickmann, A. and Ruoppolo, M.
    NATO Science for Peace and Security Series A: Chemistry and Biology (2020)
    In order to study altered molecular mechanisms representative of the damage induced by the disease in patients, two HEK293 cell models were developed. The first model was obtained with CRISPR/CAS9-based MUT gene knock out (MUT-KO). The second cell model derived from a MUT-KO cell line engineered to rescue the stable expression of MUT protein (MUT-RES). To track the quantitative changes in the global proteome of MUT-KO and MUT-RES cells, a Data Independent Acquisition mass spectrometry-based proteomic experiment was performed.Results and Discussion: Isolated methylmalonic acidemia (MMA) is a rare inherited metabolic disease of propionyl-CoA and branched-chain amino acids catabolism that affects 1 in 100,000 newborn babies. It is caused by a total or partial deficiency of methylmalonyl-CoA mutase enzymatic activity (MUT0 and MUT- subtypes, respectively). Mutations in methylmalonyl-CoA mutase (MUT) gene impair the mitochondrial conversion of methylmalonyl-CoA to succinyl-CoA, metabolized within the Krebs cycle. © 2020, Springer Nature B.V.
    view abstract10.1007/978-94-024-2041-8_15
  • Exposure of patient-derived mesenchymal stromal cells to TGFB1 supports fibrosis induction in a pediatric acute megakaryoblastic leukemia model
    Hack, T. and Bertram, S. and Blair, H. and Borger, V. and Busche, G. and Denson, L. and Fruth, E. and Giebel, B. and Heidenreich, O. and Klein-Hitpass, L. and Kollipara, L. and Sendker, S. and Sickmann, A. and Walter, C. and von Neuhoff, N. and Hanenberg, H. and Reinhardt, D. and Schneider, M. and Rasche, M.
    Molecular Cancer Research 18 (2020)
    Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL (n ¼ 5) and ML-DS (n ¼ 9). Healthy donor control MSCs (n ¼ 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an in vivo humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future. Implications: TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs. Visual Overview: 18/10/1603/F1.large.jpg. © 2020 American Association for Cancer Research.
    view abstract10.1158/1541-7786.MCR-20-0091
  • First clinical and myopathological description of a myofibrillar myopathy with congenital onset and homozygous mutation in FLNC
    Kölbel, H. and Roos, A. and van der Ven, P.F.M. and Evangelista, T. and Nolte, K. and Johnson, K. and Töpf, A. and Wilson, M. and Kress, W. and Sickmann, A. and Straub, V. and Kollipara, L. and Weis, J. and Fürst, D.O. and Schara, U.
    Human Mutation 41 (2020)
    Filamin C (encoded by the FLNC gene) is a large actin-cross-linking protein involved in shaping the actin cytoskeleton in response to signaling events both at the sarcolemma and at myofibrillar Z-discs of cross-striated muscle cells. Multiple mutations in FLNC are associated with myofibrillar myopathies of autosomal-dominant inheritance. Here, we describe for the first time a boy with congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development but no cardiac involvement associated with a homozygous FLNC mutation c.1325C>G (p.Pro442Arg). We performed ultramorphological, proteomic, and functional investigations as well as immunological studies of known marker proteins for dominant filaminopathies. We show that the mutant protein is expressed in similar quantities as the wild-type variant in control skeletal muscle fibers. The proteomic signature of quadriceps muscle is altered and ultrastructural perturbations are evident. Moreover, filaminopathy marker proteins are comparable both in our homozygous and a dominant control case (c.5161delG). Biochemical investigations demonstrate that the recombinant mutant protein is less stable and more prone to degradation by proteolytic enzymes than the wild-type variant. The unusual congenital presentation of the disease clearly demonstrates that homozygosity for mutations in FLNC severely aggravates the phenotype. © 2020 The Authors. Human Mutation published by Wiley Periodicals LLC
    view abstract10.1002/humu.24062
  • Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism
    Muscolino, E. and Schmitz, R. and Loroch, S. and Caragliano, E. and Schneider, C. and Rizzato, M. and Kim, Y.-H. and Krause, E. and Juranić Lisnić, V. and Sickmann, A. and Reimer, R. and Ostermann, E. and Brune, W.
    Nature Microbiology 5 (2020)
    Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin–proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed ‘induced protein aggregation motif’. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein–Barr virus and Kaposi’s sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
    view abstract10.1038/s41564-019-0624-1
  • Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS
    Swieringa, F. and Solari, F.A. and Pagel, O. and Beck, F. and Huang, J. and Feijge, M.A.H. and Jurk, K. and Körver-Keularts, I.M.L.W. and Mattheij, N.J.A. and Faber, J. and Pohlenz, J. and Russo, A. and Stumpel, C.T.R.M. and Schrander, D.E. and Zieger, B. and van der Meijden, P.E.J. and Zahedi, R.P. and Sickmann, A. and Heemskerk, J.W.M.
    Scientific Reports 10 (2020)
    Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia. © 2020, The Author(s).
    view abstract10.1038/s41598-020-68379-3
  • Improving identification of in-organello protein-protein interactions using an affinityenrichable, isotopically coded, and mass spectrometry-cleavable chemical crosslinker
    Makepeace, K.A.T. and Mohammed, Y. and Rudashevskaya, E.L. and Petrotchenko, E.V. and Vögtle, F.-N. and Meisinger, C. and Sickmann, A. and Borchers, C.H.
    Molecular and Cellular Proteomics 19 (2020)
    An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain. © 2020 Makepeace et al.
    view abstract10.1074/mcp.RA119.001839
  • Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome
    van Geffen, J.P. and Swieringa, F. and van Kuijk, K. and Tullemans, B.M.E. and Solari, F.A. and Peng, B. and Clemetson, K.J. and Farndale, R.W. and Dubois, L.J. and Sickmann, A. and Zahedi, R.P. and Ahrends, R. and Biessen, E.A.L. and Sluimer, J.C. and Heemskerk, J.W.M. and Kuijpers, M.J.E.
    Scientific Reports 10 (2020)
    Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe−/− and Ldlr−/− mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe−/− mice). Bone marrow from wild-type or Ldlr−/− mice was transplanted into irradiated Ldlr−/− recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe−/− and Ldlr−/− mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype. © 2020, The Author(s).
    view abstract10.1038/s41598-020-78522-9
  • Mitochondrial CLPP2 Assists Coordination and Homeostasis of Respiratory Complexes1[OPEN]
    Petereit, J. and Duncan, O. and Murcha, M.W. and Fenske, A.R. and Cincu, E. and Cahn, J. and PruzinskÃi, A. and Ivanova, A. and Kollipara, L. and Wortelkamp, S. and Sickmann, A. and Lee, J. and Lister, R. and Millar, A.H. and Huang, S.
    Plant Physiology 184 (2020)
    Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear- A nd mitochondrial-encoded subunits for complex assembly in mitochondria await discovery. We generated knockout lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis (Arabidopsis thaliana). Mutants showed a higher abundance of transcripts from mitochondrial genes encoding oxidative phosphorylation protein complexes, whereas nuclear genes encoding other subunits of the same complexes showed no change in transcript abundance. By contrast, the protein abundance of specific nuclear-encoded subunits in oxidative phosphorylation complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Complexes with subunits mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import and function of complex I revealed that while function was retained, protein homeostasis was disrupted, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and homeostasis of protein complexes encoded across mitochondrial and nuclear genomes. © 2020 American Society of Plant Biologists. All rights reserved.
    view abstract10.1104/PP.20.00136
  • Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome
    Sims, M.C. and Mayer, L. and Collins, J.H. and Bariana, T.K. and Megy, K. and Lavenu-Bombled, C. and Seyres, D. and Kollipara, L. and Burden, F.S. and Greene, D. and Lee, D. and Rodriguez-Romera, A. and Alessi, M.-C. and Astle, W.J. and Bahou, W.F. and Bury, L. and Chalmers, E. and Da Silva, R. and de Candia, E. and Deevi, S.V.V. and Farrow, S. and Gomez, K. and Grassi, L. and Greinacher, A. and Gresele, P. and Hart, D. and Hurtaud, M.-F. and Kelly, A.M. and Kerr, R. and Le Quellec, S. and Leblanc, T. and Leinøe, E.B. and Mapeta, R. and McKinney, H. and Michelson, A.D. and Morais, S. and Nugent, D. and Papadia, S. and Park, S.J. and Pasi, J. and Podda, G.M. and Poon, M.-C. and Reed, R. and Sekhar, M. and Shalev, H. and Sivapalaratnam, S. and Steinberg-Shemer, O. and Stephens, J.C. and Tait, R.C. and Turro, E. and Wu, J.K.M. and Zieger, B. and Kuijpers, T.W. and Whetton, A.D. and Sickmann, A. and Freson, K. and Downes, K. and Erber, W.N. and Frontini, M. and Nurden, P. and Ouwehand, W.H. and Favier, R. and Guerrero, J.A. and NIHR BioResource
    Blood 136 (2020)
    Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet a-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease. © 2020 by The American Society of Hematology.
    view abstract10.1182/BLOOD.2019004776
  • Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth
    Märker, R. and Blank-Landeshammer, B. and Beier-Rosberger, A. and Sickmann, A. and Kück, U.
    Molecular Microbiology 113 (2020)
    The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex. © 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd
    view abstract10.1111/mmi.14475
  • Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics
    Kopczynski, D. and Hentschel, A. and Coman, C. and Schebb, N.H. and Hornemann, T. and Mashek, D.G. and Hartung, N.M. and Shevchuk, O. and Schött, H.-F. and Lorenz, K. and Torta, F. and Burla, B. and Zahedi, R.P. and Sickmann, A. and Kreutz, M.R. and Ejsing, C.S. and Medenbach, J. and Ahrends, R.
    Analytical Chemistry 92 (2020)
    We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms. © 2020 American Chemical Society.
    view abstract10.1021/acs.analchem.0c02793
  • Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies
    Xuan, Y. and Bateman, N.W. and Gallien, S. and Goetze, S. and Zhou, Y. and Navarro, P. and Hu, M. and Parikh, N. and Hood, B.L. and Conrads, K.A. and Loosse, C. and Kitata, R.B. and Piersma, S.R. and Chiasserini, D. and Zhu, H. and Hou, G. and Tahir, M. and Macklin, A. and Khoo, A. and Sun, X. and Crossett, B. and Sickmann, A. and Chen, Y.-J. and Jimenez, C.R. and Zhou, H. and Liu, S. and Larsen, M.R. and Kislinger, T. and Chen, Z. and Parker, B.L. and Cordwell, S.J. and Wollscheid, B. and Conrads, T.P.
    Nature Communications 11 (2020)
    Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality. © 2020, The Author(s).
    view abstract10.1038/s41467-020-18904-9
  • Targeted Approach to Distinguish and Determine Absolute Levels of GDF8 and GDF11 in Mouse Serum
    Camparini, L. and Kollipara, L. and Sinagra, G. and Loffredo, F.S. and Sickmann, A. and Shevchuk, O.
    Proteomics 20 (2020)
    Growth differentiation factor 11 (GDF11) is a TGF-β superfamily circulating factor that regulates cardiomyocyte size in rodents, sharing 90% amino acid sequence identity in the active domains with myostatin (GDF8)—the major determinant of skeletal muscle mass. Conflicting data on age-related changes in circulating levels have been reported mainly due to the lack of specific detection methods. More recently, liquid chromatography tandem mass spectrometry (LC-MS/MS) based assay showed that the circulating levels of GDF11 do not change significantly throughout human lifespan, but GDF8 levels decrease with aging in men. Here a novel detection method is demonstrated based on parallel reaction monitoring LC-MS/MS assay combined with immunoprecipitation to reliably distinguish GDF11 and GDF8 as well as determine their endogenous levels in mouse serum. The data indicate that both GDF11 and GDF8 circulating levels significantly decline with aging in female mice. © 2020 The Authors. Proteomics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/pmic.201900104
  • The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes
    Stein, V. and Blank-Landeshammer, B. and Müntjes, K. and Märker, R. and Teichert, I. and Feldbrügge, M. and Sickmann, A. and Kück, U.
    PLoS Genetics 16 (2020)
    The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343. served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phosphodeficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phosphodeficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth. © 2020 Stein et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    view abstract10.1371/journal.pgen.1008819
  • A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells
    Nguyen, C.D.L. and Malchow, S. and Reich, S. and Steltgens, S. and Shuvaev, K.V. and Loroch, S. and Lorenz, C. and Sickmann, A. and Knobbe-Thomsen, C.B. and Tews, B. and Medenbach, J. and Ahrends, R.
    Scientific Reports 9 (2019)
    Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines. © 2019, The Author(s).
    view abstract10.1038/s41598-019-45237-5
  • Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes
    Rodziewicz, P. and Loroch, S. and Marczak and Sickmann, A. and Kayser, O.
    Plant Science 284 (2019)
    Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes. © 2019 Elsevier B.V.
    view abstract10.1016/j.plantsci.2019.04.008
  • Combination of proteogenomics with peptide de novo sequencing identifies new genes and hidden posttranscriptional modifications
    Blank-Landeshammer, B. and Teichert, I. and Märker, R. and Nowrousian, M. and Kück, U. and Sickmann, A.
    mBio 10 (2019)
    Proteogenomics combines proteomics, genomics, and transcriptomics and has considerably improved genome annotation in poorly investigated phylogenetic groups for which homology information is lacking. Furthermore, it can be advantageous when reinvestigating well-annotated genomes. Here, we applied an advanced proteogenomics approach, combining standard proteogenomics with peptide de novo sequencing, to refine annotation of the well-studied model fungus Sordaria macrospora. We investigated samples from different developmental and physiological conditions, resulting in the detection of 104 so-far hidden proteins and annotation changes in 575 genes, including 389 splice site refinements. Significantly, our approach provides peptide-level evidence for 113 single-amino-acid variations and 15 C-terminal protein elongations originating from A-to-I RNA editing, a phenomenon recently detected in fungi. Coexpression and phylostratigraphic analysis of the refined proteome suggest that new functions in evolutionarily young genes correlate with distinct developmental stages. In conclusion, our advanced proteogenomics approach supports and promotes functional studies of fungal model systems. © 2019 Blank-Landeshammer et al.
    view abstract10.1128/mBio.02367-19
  • Investigating the Role of Mitochondria in Type 2 Diabetes – Lessons from Lipidomics and Proteomics Studies of Skeletal Muscle and Liver
    Kappler, L. and Kollipara, L. and Lehmann, R. and Sickmann, A.
    Advances in Experimental Medicine and Biology 1158 (2019)
    Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondrial lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre-analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed. © 2019, Springer Nature Singapore Pte Ltd.
    view abstract10.1007/978-981-13-8367-0_9
  • Linking bioenergetic function of mitochondria to tissue-specific molecular fingerprints
    Kappler, L. and Hoene, M. and Hu, C. and von Toerne, C. and Li, J. and Bleher, D. and Hoffmann, C. and Böhm, A. and Kollipara, L. and Zischka, H. and Königsrainer, A. and Häring, H.-U. and Peter, A. and Xu, G. and Sickmann, A. and Hauck, S.M. and Weigert, C. and Lehmann, R.
    American Journal of Physiology - Endocrinology and Metabolism 317 (2019)
    Mitochondria are dynamic organelles with diverse functions in tissues such as liver and skeletal muscle. To unravel the mitochondrial contribution to tissue-specific physiology, we performed a systematic comparison of the mitochondrial proteome and lipidome of mice and assessed the consequences hereof for respiration. Liver and skeletal muscle mitochondrial protein composition was studied by data-independent ultra-high-performance (UHP)LC-MS/ MS-proteomics, and lipid profiles were compared by UHPLC-MS/MS lipidomics. Mitochondrial function was investigated by high-resolution respirometry in samples from mice and humans. Enzymes of pyruvate oxidation as well as several subunits of complex I, III, and ATP synthase were more abundant in muscle mitochondria. Muscle mitochondria were enriched in cardiolipins associated with higher oxidative phosphorylation capacity and flexibility, in particular CL(18:2)4 and 22:6-containing cardiolipins. In contrast, protein equipment of liver mitochondria indicated a shuttling of complex I substrates toward gluconeogenesis and ketogenesis and a higher preference for electron transfer via the flavoprotein quinone oxi-doreductase pathway. Concordantly, muscle and liver mitochondria showed distinct respiratory substrate preferences. Muscle respired significantly more on the complex I substrates pyruvate and glutamate, whereas in liver maximal respiration was supported by complex II substrate succinate. This was a consistent finding in mouse liver and skeletal muscle mitochondria and human samples. Muscle mitochondria are tailored to produce ATP with a high capacity for complex I-linked substrates. Liver mitochondria are more connected to biosyn-thetic pathways, preferring fatty acids and succinate for oxidation. The physiologic diversity of mitochondria may help to understand tissue-specific disease pathologies and to develop therapies targeting mitochondrial function. © 2019 the American Physiological Society.
    view abstract10.1152/ajpendo.00088.2019
  • O-GlcNAcylation of Histone Deacetylase 4 Protects the Diabetic Heart From Failure
    Kronlage, M. and Dewenter, M. and Grosso, J. and Fleming, T. and Oehl, U. and Lehmann, L.H. and Falcão-Pires, I. and Leite-Moreira, A.F. and Volk, N. and Gröne, H.-J. and Müller, O.J. and Sickmann, A. and Katus, H.A. and Backs, J.
    Circulation 140 (2019)
    BACKGROUND: Worldwide, diabetes mellitus and heart failure represent frequent comorbidities with high socioeconomic impact and steadily growing incidence, calling for a better understanding of how diabetic metabolism promotes cardiac dysfunction. Paradoxically, some glucose-lowering drugs have been shown to worsen heart failure, raising the question of how glucose mediates protective versus detrimental cardiac signaling. Here, we identified a histone deacetylase 4 (HDAC4) subdomain as a molecular checkpoint of adaptive and maladaptive signaling in the diabetic heart. METHODS: A conditional HDAC4 allele was used to delete HDAC4 specifically in cardiomyocytes (HDAC4-knockout). Mice were subjected to diabetes mellitus either by streptozotocin injections (type 1 diabetes mellitus model) or by crossing into mice carrying a leptin receptor mutation (db/db; type 2 diabetes mellitus model) and monitored for remodeling and cardiac function. Effects of glucose and the posttranslational modification by β-linked N-acetylglucosamine (O-GlcNAc) on HDAC4 were investigated in vivo and in vitro by biochemical and cellular assays. RESULTS: We show that the cardio-protective N-terminal proteolytic fragment of HDAC4 is enhanced in vivo in patients with diabetes mellitus and mouse models, as well as in vitro under high-glucose and high-O-GlcNAc conditions. HDAC4-knockout mice develop heart failure in models of type 1 and type 2 diabetes mellitus, whereas wild-type mice do not develop clear signs of heart failure, indicating that HDAC4 protects the diabetic heart. Reexpression of the N-terminal fragment of HDAC4 prevents HDAC4-dependent diabetic cardiomyopathy. Mechanistically, the posttranslational modification of HDAC4 at serine (Ser)-642 by O-GlcNAcylation is an essential step for production of the N-terminal fragment of HDAC4, which was attenuated by Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation at Ser-632. Preventing O-GlcNAcylation at Ser-642 not only entirely precluded production of the N-terminal fragment of HDAC4 but also promoted Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation at Ser-632, pointing to a mutual posttranslational modification cross talk of (cardio-detrimental) phosphorylation at Ser-632 and (cardio-protective) O-GlcNAcylation at Ser-642. CONCLUSIONS: In this study, we found that O-GlcNAcylation of HDAC4 at Ser-642 is cardio-protective in diabetes mellitus and counteracts pathological Ca2+/calmodulin-dependent protein kinase II signaling. We introduce a molecular model explaining how diabetic metabolism possesses important cardio-protective features besides its known detrimental effects. A deeper understanding of the here-described posttranslational modification cross talk may lay the groundwork for the development of specific therapeutic concepts to treat heart failure in the context of diabetes mellitus.
    view abstract10.1161/CIRCULATIONAHA.117.031942
  • Phosphorylation of the Bruchpilot N-terminus in Drosophila unlocks axonal transport of active zone building blocks
    Driller, J.H. and Tzkendorf, J.L. and Depner, H. and Siebert, M. and Kuropka, B. and Weise, C. and Piao, C. and Petzoldt, A.G. and Lehmann, M. and Stelzl, U. and Zahedi, R. and Sickmann, A. and Freund, C. and Sigrist, S.J. and Wahl, M.C.
    Journal of Cell Science 132 (2019)
    Protein scaffolds at presynaptic active zone membranes control information transfer at synapses. For scaffold biogenesis and maintenance, scaffold components must be safely transported along axons. A spectrum of kinases has been suggested to control transport of scaffold components, but direct kinase-substrate relationships and operational principles steering phosphorylationdependent active zone protein transport are presently unknown. Here, we show that extensive phosphorylation of a 150-residue unstructured region at the N-terminus of the highly elongated Bruchpilot (BRP) active zone protein is crucial for ordered active zone precursor transport in Drosophila. Point mutations that block SRPK79D kinase-mediated phosphorylation of the BRP N-terminus interfered with axonal transport, leading to BRP-positive axonal aggregates that also contain additional active zone scaffold proteins. Axonal aggregates formed only in the presence of nonphosphorylatable BRP isoforms containing the SRPK79D-targeted N-terminal stretch. We assume that specific active zone proteins are pre-assembled in transport packages and are thus co-transported as functional scaffold building blocks. Our results suggest that transient post-translational modification of a discrete unstructured domain of the master scaffold component BRP blocks oligomerization of these building blocks during their long-range transport. © 2019. Published by The Company of Biologists Ltd.
    view abstract10.1242/jcs.225151
  • Proteogenomics of colorectal cancer liver metastases: Complementing precision oncology with phenotypic data
    Blank-Landeshammer, B. and Richard, V.R. and Mitsa, G. and Marques, M. and Leblanc, A. and Kollipara, L. and Feldmann, I. and Du Tertre, M.C. and Gambaro, K. and McNamara, S. and Spatz, A. and Zahedi, R.P. and Sickmann, A. and Batist, G. and Borchers, C.H.
    Cancers 11 (2019)
    Hotspot testing for activating KRAS mutations is used in precision oncology to select colorectal cancer (CRC) patients who are eligible for anti-EGFR treatment. However, even for KRASwildtype tumors anti-EGFR response rates are <30%, while mutated-KRAS does not entirely rule out response, indicating the need for improved patient stratification. We performed proteogenomic phenotyping of KRASwildtype and KRASG12V CRC liver metastases (mCRC). Among >9000 proteins we detected considerable expression changes including numerous proteins involved in progression and resistance in CRC. We identified peptides representing a number of predicted somatic mutations, including KRASG12V. For eight of these, we developed a multiplexed parallel reaction monitoring (PRM) mass spectrometry assay to precisely quantify the mutated and canonical protein variants. This allowed phenotyping of eight mCRC tumors and six paired healthy tissues, by determining mutation rates on the protein level. Total KRAS expression varied between tumors (0.47–1.01 fmol/μg total protein) and healthy tissues (0.13–0.64 fmol/μg). In KRASG12V-mCRC, G12Vmutation levels were 42–100%, while one patient had only 10% KRASG12V but 90% KRASwildtype. This might represent a missed therapeutic opportunity: Based on hotspot sequencing, the patient was excluded from anti-EGFR treatment and instead received chemotherapy, while PRM-based tumorphenotyping indicates the patient might have benefitted from anti-EGFR therapy. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/cancers11121907
  • Short peptides with uncleavable peptide bond mimetics as photoactivatable caspase-3 inhibitors
    Van Kersavond, T. and Konopatzki, R. and Chakrabarty, S. and Blank-Landeshammer, B. and Sickmann, A. and Verhelst, S.H.L.
    Molecules 24 (2019)
    Chemical probes that covalently interact with proteases have found increasing use for the study of protease function and localization. The design and synthesis of such probes is still a bottleneck, as the strategies to target different families are highly diverse. We set out to design and synthesize chemical probes based on protease substrate specificity with inclusion of an uncleavable peptide bond mimic and a photocrosslinker for covalent modification of the protease target. With caspase-3 as a model target protease, we designed reduced amide and triazolo peptides as substrate mimetics, whose sequences can be conveniently constructed by modified solid phase peptide synthesis. We found that these probes inhibited the caspase-3 activity, but did not form a covalent bond. It turned out that the reduced amide mimics, upon irradiation with a benzophenone as photosensitizer, are oxidized and form low concentrations of peptide aldehydes, which then act as inhibitors of caspase-3. This type of photoactivation may be utilized in future photopharmacology experiments to form protease inhibitors at a precise time and location. © 2019 by the authors
    view abstract10.3390/molecules24010206
  • Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range
    Ostermann, E. and Loroch, S. and Qian, Z. and Sickmann, A. and Wiebusch, L. and Brune, W.
    PLoS Pathogens 14 (2018)
    Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells. © 2018 Ostermann et al.
    view abstract10.1371/journal.ppat.1007481
  • Advanced tools for the analysis of protein phosphorylation in yeast mitochondria
    Walter, C. and Gonczarowska-Jorge, H. and Sickmann, A. and Zahedi, R.P. and Meisinger, C. and Schmidt, O.
    Analytical Biochemistry 554 (2018)
    The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn2+-Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods. © 2018
    view abstract10.1016/j.ab.2018.05.022
  • Analysis of new growth promoting black market products
    Krug, O. and Thomas, A. and Malerød-Fjeld, H. and Dehnes, Y. and Laussmann, T. and Feldmann, I. and Sickmann, A. and Thevis, M.
    Growth Hormone and IGF Research 41 (2018)
    Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3–6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration. © 2018
    view abstract10.1016/j.ghir.2018.05.001
  • Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry
    Mnatsakanyan, R. and Shema, G. and Basik, M. and Batist, G. and Borchers, C.H. and Sickmann, A. and Zahedi, R.P.
    Expert Review of Proteomics 15 (2018)
    Introduction: Numerous diseases are caused by changes in post-translational modifications (PTMs). Therefore, the number of clinical proteomics studies that include the analysis of PTMs is increasing. Combining complementary information—for example changes in protein abundance, PTM levels, with the genome and transcriptome (proteogenomics)—holds great promise for discovering important drivers and markers of disease, as variations in copy number, expression levels, or mutations without spatial/functional/isoform information is often insufficient or even misleading. Areas covered: We discuss general considerations, requirements, pitfalls, and future perspectives in applying PTM-centric proteomics to clinical samples. This includes samples obtained from a human subject, for instance (i) bodily fluids such as plasma, urine, or cerebrospinal fluid, (ii) primary cells such as reproductive cells, blood cells, and (iii) tissue samples/biopsies. Expert commentary: PTM-centric discovery proteomics can substantially contribute to the understanding of disease mechanisms by identifying signatures with potential diagnostic or even therapeutic relevance but may require coordinated efforts of interdisciplinary and eventually multi-national consortia, such as initiated in the cancer moonshot program. Additionally, robust and standardized mass spectrometry (MS) assays—particularly targeted MS, MALDI imaging, and immuno-MALDI—may be transferred to the clinic to improve patient stratification for precision medicine, and guide therapies. © 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
    view abstract10.1080/14789450.2018.1483340
  • Disentangling thermal stress responses in a reef-calcifier and its photosymbionts by shotgun proteomics
    Stuhr, M. and Blank-Landeshammer, B. and Reymond, C.E. and Kollipara, L. and Sickmann, A. and Kucera, M. and Westphal, H.
    Scientific Reports 8 (2018)
    The proliferation of key marine ecological engineers and carbonate producers often relies on their association with photosymbiotic algae. Evaluating stress responses of these organisms is important to predict their fate under future climate projections. Physiological approaches are limited in their ability to resolve the involved molecular mechanisms and attribute stress effects to the host or symbiont, while probing and partitioning of proteins cannot be applied in organisms where the host and symbiont are small and cannot be physically separated. Here we apply a label-free quantitative proteomics approach to detect changes of proteome composition in the diatom-bearing benthic foraminifera Amphistegina gibbosa experimentally exposed to three thermal-stress scenarios. We developed a workflow for protein extraction from less than ten specimens and simultaneously analysed host and symbiont proteomes. Despite little genomic data for the host, 1,618 proteins could be partially assembled and assigned. The proteomes revealed identical pattern of stress response among stress scenarios as that indicated by physiological measurements, but allowed identification of compartment-specific stress reactions. In the symbiont, stress-response and proteolysis-related proteins were up regulated while photosynthesis-related proteins declined. In contrast, host homeostasis was maintained through chaperone up-regulation associated with elevated proteosynthesis and proteolysis, and the host metabolism shifted to heterotrophy. © 2018 The Author(s).
    view abstract10.1038/s41598-018-21875-z
  • Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets
    Makhoul, S. and Walter, E. and Pagel, O. and Walter, U. and Sickmann, A. and Gambaryan, S. and Smolenski, A. and Zahedi, R.P. and Jurk, K.
    Nitric Oxide - Biology and Chemistry 76 (2018)
    Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. © 2018 Elsevier Inc.
    view abstract10.1016/j.niox.2018.03.008
  • Identification of key lipids critical for platelet activation by comprehensive analysis of the platelet lipidome
    Peng, B. and Geue, S. and Coman, C. and Münzer, P. and Kopczynski, D. and Has, C. and Hoffmann, N. and Manke, M.-C. and Lang, F. and Sickmann, A. and Gawaz, M. and Borst, O. and Ahrends, R.
    Blood 132 (2018)
    Platelet integrity and function critically depend on lipid composition. However, the lipid inventory in platelets was hitherto not quantified. Here, we examined the lipidome of murine platelets using lipid-category tailored protocols on a quantitative lipidomics platform. We could show that the platelet lipidome comprises almost 400 lipid species and covers a concentration range of 7 orders of magnitude. A systematic comparison of the lipidomics network in resting and activated murine platelets, validated in human platelets, revealed that <20% of the platelet lipidome is changed upon activation, involving mainly lipids containing arachidonic acid. Sphingomyelin phosphodiesterase-1 (Smpd1) deficiency resulted in a very specific modulation of the platelet lipidome with an order of magnitude upregulation of lysosphingomyelin (SPC), and subsequent modification of platelet activation and thrombus formation. In conclusion, this first comprehensive quantitative lipidomic analysis of platelets sheds light on novel mechanisms important for platelet function, and has therefore the potential to open novel diagnostic and therapeutic opportunities. © 2018 by The American Society of Hematology.
    view abstract10.1182/blood-2017-12-822890
  • Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation
    Bruning, U. and Morales-Rodriguez, F. and Kalucka, J. and Goveia, J. and Taverna, F. and Queiroz, K.C.S. and Dubois, C. and Cantelmo, A.R. and Chen, R. and Loroch, S. and Timmerman, E. and Caixeta, V. and Bloch, K. and Conradi, L.-C. and Treps, L. and Staes, A. and Gevaert, K. and Tee, A. and Dewerchin, M. and Semenkovich, C.F. and Impens, F. and Schilling, B. and Verdin, E. and Swinnen, J.V. and Meier, J.L. and Kulkarni, R.A. and Sickmann, A. and Ghesquière, B. and Schoonjans, L. and Li, X. and Mazzone, M. and Carmeliet, P.
    Cell Metabolism 28 (2018)
    The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASN KD ) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at &gt;10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASN KD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASN KD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade. © 2018 Elsevier Inc. Bruning et al. report that blocking fatty acid synthase (FASN) in endothelial cells (ECs) reduces angiogenesis by impairing EC proliferation. Mechanistically, FASN inhibition elevates the malonyl-CoA substrate pool, thereby increasing post-translational malonylation of mTOR and decreasing the pro-angiogenic mTORC1 activity. © 2018 Elsevier Inc.
    view abstract10.1016/j.cmet.2018.07.019
  • Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures
    Rodrigues, R.M. and Kollipara, L. and Chaudhari, U. and Sachinidis, A. and Zahedi, R.P. and Sickmann, A. and Kopp-Schneider, A. and Jiang, X. and Keun, H. and Hengstler, J. and Oorts, M. and Annaert, P. and Hoeben, E. and Gijbels, E. and De Kock, J. and Vanhaecke, T. and Rogiers, V. and Vinken, M.
    Archives of Toxicology 92 (2018)
    Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.
    view abstract10.1007/s00204-018-2214-z
  • Platelet proteomics: from discovery to diagnosis
    Looße, C. and Swieringa, F. and Heemskerk, J.W.M. and Sickmann, A. and Lorenz, C.
    Expert Review of Proteomics 15 (2018)
    Introduction: Platelets are the smallest cells within the circulating blood with key roles in physiological hemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signaling cascades. Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications. Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signaling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group.
    view abstract10.1080/14789450.2018.1480111
  • Simple, scalable, and ultrasensitive tip-based identification of protease substrates
    Shema, G. and Nguyen, M.T.N. and Solari, F.A. and Loroch, S. and Venne, A.S. and Kollipara, L. and Sickmann, A. and Verhelst, S.H.L. and Zahedi, R.P.
    Molecular and Cellular Proteomics 17 (2018)
    Proteases are in the center of many diseases, and consequently, proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N termini. Methods such as combined fractional diagonal chromatography (COFRADIC)1 and, later, terminal amine isotopic labeling of substrates (TAILS) have revealed numerous insights into protease substrates and consensus motifs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on charge-based fractional diagonal chromatography (ChaFRADIC) and requiring only well-established protein chemistry and a pipette tip. Using iTRAQ-8-plex, we quantified on average 2,073 52 unique N-terminal peptides from only 4.3 g per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow working with clinical samples such as needle biopsies in the future. We applied our method to study the dynamics of staurosporine-induced apoptosis. Our data demonstrate an orchestrated regulation of specific pathways after 1.5 h, 3 h, and 6 h of treatment, with many important players of homeostasis targeted already after 1.5 h. We additionally observed an early multilevel modulation of the splicing machinery both by proteolysis and phosphorylation. This may reflect the known role of alternative splicing variants for a variety of apoptotic genes, which seems to be a driving force of staurosporine-induced apoptosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
    view abstract10.1074/mcp.TIR117.000302
  • Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea
    Zweerink, S. and Kallnik, V. and Ninck, S. and Nickel, S. and Verheyen, J. and Blum, M. and Wagner, A. and Feldmann, I. and Sickmann, A. and Albers, S.-V. and Bräsen, C. and Kaschani, F. and Siebers, B. and Kaiser, M.
    Nature Communications 8 (2017)
    Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Here, we demonstrate that in vivo activity-based protein profiling (ABPP) may be used to sensitively detect either native or heterogeneously expressed active enzymes in living archaea even under these extreme conditions. In combination with the development of a genetically engineered archaeal screening strain, ABPP can furthermore be used in functional enzyme screenings from (meta)genome samples. We anticipate that our ABPP approach may therefore find application in basic archaeal research but also in the discovery of novel enzymes from (meta)genome libraries. © 2017 The Author(s).
    view abstract10.1038/ncomms15352
  • Alterations of the platelet proteome in type I Glanzmann thrombasthenia caused by different homozygous delG frameshift mutations in ITGA2B
    Loroch, S. and Trabold, K. and Gambaryan, S. and Reiß, C. and Schwierczek, K. and Fleming, I. and Sickmann, A. and Behnisch, W. and Zieger, B. and Zahedi, R.P. and Walter, U. and Jurk, K.
    Thrombosis and Haemostasis 117 (2017)
    Glanzmann thrombasthenia (GT) is one of the best characterised inherited platelet function disorders but global platelet proteome has not been determined in these patients. We investigated the proteome and function of platelets from two patients with type I GT, caused by different homozygous ITGA2b mutations, from family members and unrelated controls. The global proteome of highly purified washed platelets was quantified by liquid chromatography-mass spectrometry (LC-MS) and targeted MS-methods. Platelet function was analysed by flow cytometry, light transmission aggregometry and flow-based assays. Platelets from GT patients showed less than 5 % relative levels of the integrin subunit αIIb and 5-9 % fibrinogen compared to controls. These patients demonstrated loss of αIIbβ3-dependent platelet function, but normal platelet granule secretion induced by physiological agonists. Platelets from heterozygous family members of a patient expressed 50-60 % of control αIIb levels which were sufficient for normal αIIbβ3-dependent platelet function. Studying type I GT as model disease we established quantitative LC-MS to detect and clearly distinguish normal platelets, platelets from GT heterozygotes and platelets from GT patients. Diminished levels of factor XIIIB chain, plasminogen and carboxypeptidase 2B were identified in thrombasthenic platelets. Additionally, GT platelets showed up to 2.5-fold increased levels of FcγRIIA and laminin-α4 chain. Elevated levels of platelet FcγRIIA was associated with increased CD63-surface expression after FcγRIIAcrosslinking in one GT-patient which might present a compensatory mechanism of platelet activation in GT. We demonstrate that quantitative LC-MS based proteomics is suitable to validate known but also to identify previously unknown protein level changes of dysfunctional platelets. © Schattauer 2017.
    view abstract10.1160/TH16-07-0515
  • Combining de Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics
    Blank-Landeshammer, B. and Kollipara, L. and Biß, K. and Pfenninger, M. and Malchow, S. and Shuvaev, K. and Zahedi, R.P. and Sickmann, A.
    Journal of Proteome Research 16 (2017)
    Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion. © 2017 American Chemical Society.
    view abstract10.1021/acs.jproteome.7b00198
  • Comparison and Evaluation of Clustering Algorithms for Tandem Mass Spectra
    Rieder, V. and Schork, K.U. and Kerschke, L. and Blank-Landeshammer, B. and Sickmann, A. and Rahnenführer, J.
    Journal of Proteome Research 16 (2017)
    In proteomics, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established for identifying peptides and proteins. Duplicated spectra, that is, multiple spectra of the same peptide, occur both in single MS/MS runs and in large spectral libraries. Clustering tandem mass spectra is used to find consensus spectra, with manifold applications. First, it speeds up database searches, as performed for instance by Mascot. Second, it helps to identify novel peptides across species. Third, it is used for quality control to detect wrongly annotated spectra. We compare different clustering algorithms based on the cosine distance between spectra. CAST, MS-Cluster, and PRIDE Cluster are popular algorithms to cluster tandem mass spectra. We add well-known algorithms for large data sets, hierarchical clustering, DBSCAN, and connected components of a graph, as well as the new method N-Cluster. All algorithms are evaluated on real data with varied parameter settings. Cluster results are compared with each other and with peptide annotations based on validation measures such as purity. Quality control, regarding the detection of wrongly (un)annotated spectra, is discussed for exemplary resulting clusters. N-Cluster proves to be highly competitive. All clustering results benefit from the so-called DISMS2 filter that integrates additional information, for example, on precursor mass. © 2017 American Chemical Society.
    view abstract10.1021/acs.jproteome.7b00427
  • Computational proteomics tools for identification and quality control
    Kopczynski, D. and Sickmann, A. and Ahrends, R.
    Journal of Biotechnology (2017)
    Computational proteomics is a constantly growing field to support end users with powerful and reliable tools for performing several computational steps within an analytics workflow for proteomics experiments. Typically, after capturing with a mass spectrometer, the proteins have to be identified and quantified. After certain follow-up analyses, an optional targeted approach is suitable for validating the results. The de.NBI (German network for bioinformatics infrastructure) service center in Dortmund provides several software applications and platforms as services to meet these demands. In this work, we present our tools and services, which is the combination of SearchGUI and PeptideShaker. SearchGUI is a managing tool for several search engines to find peptide spectra matches for one or more complex MS2 measurements. PeptideShaker combines all matches and creates a consensus list of identified proteins providing statistical confidence measures. In a next step, we are planning to release a web service for protein identification containing both tools. This system will be designed for high scalability and distributed computing using solutions like the Docker container system among others. As an additional service, we offer a web service oriented database providing all necessary high-quality and high-resolution data for starting targeted proteomics analyses. The user can easily select proteins of interest, review the according spectra and download both protein sequences and spectral library. All systems are designed to be intuitively and user-friendly operable. © 2017 Elsevier B.V.
    view abstract10.1016/j.jbiotec.2017.06.1199
  • Deciphering lymphoma pathogenesis via state-of-the-art mass spectrometry-based quantitative proteomics
    Psatha, K. and Kollipara, L. and Voutyraki, C. and Divanach, P. and Sickmann, A. and Rassidakis, G.Z. and Drakos, E. and Aivaliotis, M.
    Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 1047 (2017)
    Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenesis of lymphoma has incremental value in deciphering the heterogeneity in complex deregulated molecular mechanisms/pathways of the lymphoma entities, implementing the current diagnostic and therapeutic strategies. Essential global, targeted and functional differential proteomics analyses although still evolving, have been successfully implemented to shed light on lymphoma pathogenesis to discover and explore the role of potential lymphoma biomarkers and drug targets. This review aims to outline and appraise the present status of MS-based quantitative proteomic approaches in lymphoma research, introducing the current state-of-the-art MS-based proteomic technologies, the opportunities they offer in biological discovery in human lymphomas and the related limitation issues arising from sample preparation to data evaluation. It is a synopsis containing information obtained from recent research articles, reviews and public proteomics repositories (PRIDE). We hope that this review article will aid, assimilate and assess all the information aiming to accelerate the development and validation of diagnostic, prognostic or therapeutic targets for an improved and empowered clinical proteomics application in lymphomas in the nearby future. © 2016 Elsevier B.V.
    view abstract10.1016/j.jchromb.2016.11.005
  • DISMS2: A flexible algorithm for direct proteome- Wide distance calculation of LC-MS/MS runs
    Rieder, V. and Blank-Landeshammer, B. and Stuhr, M. and Schell, T. and Biß, K. and Kollipara, L. and Meyer, A. and Pfenninger, M. and Westphal, H. and Sickmann, A. and Rahnenführer, J.
    BMC Bioinformatics 18 (2017)
    Background: The classification of samples on a molecular level has manifold applications, from patient classification regarding cancer treatment to phylogenetics for identifying evolutionary relationships between species. Modern methods employ the alignment of DNA or amino acid sequences, mostly not genome-wide but only on selected parts of the genome. Recently proteomics-based approaches have become popular. An established method for the identification of peptides and proteins is liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, protein sequences from MS/MS spectra are identified by means of database searches, given samples with known genome-wide sequence information, then sequence based methods are applied. Alternatively, de novo peptide sequencing algorithms annotate MS/MS spectra and deduce peptide/protein information without a database. A newer approach independent of additional information is to directly compare unidentified tandem mass spectra. The challenge then is to compute the distance between pairwise MS/MS runs consisting of thousands of spectra. Methods: We present DISMS2, a new algorithm to calculate proteome-wide distances directly from MS/MS data, extending the algorithm compareMS2, an approach that also uses a spectral comparison pipeline. Results: Our new more flexible algorithm, DISMS2, allows for the choice of the spectrum distance measure and includes different spectra preprocessing and filtering steps that can be tailored to specific situations by parameter optimization. Conclusions: DISMS2 performs well for samples from species with and without database annotation and thus has clear advantages over methods that are purely based on database search. © 2017 The Author(s).
    view abstract10.1186/s12859-017-1514-2
  • Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC)
    Tinnefeld, V. and Venne, A.S. and Sickmann, A. and Zahedi, R.P.
    Journal of Proteome Research 16 (2017)
    Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. We present a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). On the basis of two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture, and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible, and independent of the cross-linking chemistry and cross-linker properties. © 2016 American Chemical Society.
    view abstract10.1021/acs.jproteome.6b00587
  • Intersection of Proteomics and Genomics to "Solve the Unsolved" in Rare Disorders such as Neurodegenerative and Neuromuscular Diseases
    Roos, A. and Thompson, R. and Horvath, R. and Lochmüller, H. and Sickmann, A.
    Proteomics - Clinical Applications (2017)
    Despite recent rapid advances in sequencing technologies, a significant proportion of patients with rare genetic disorders do not receive a genetic diagnosis after exhaustive testing, and even fewer have a potential therapeutic target identified. Taking rare neuromuscular and neurodegenerative disorders as a paradigm that can be extended to other rare Mendelian disorders, this viewpoint explores the opportunities that are brought about by the integration of genomics and proteomics, as well as the limitations and remaining challenges of this newly emerging field of proteogenomics. The relevance of combining proteomic findings with genetic results for diagnosis and gene discovery is illustrated, highlighting the insights the combined analysis provides into the underlying biology and aetiology as well as the limitations of the experimental techniques. A final discussion focuses on the importance of mechanisms to enable the sharing, reuse, and analysis of source experimental data and describes some of the international initiatives that are making progress in this area. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/prca.201700073
  • Landscape of submitochondrial protein distribution
    Vögtle, F.-N. and Burkhart, J.M. and Gonczarowska-Jorge, H. and Kücükköse, C. and Taskin, A.A. and Kopczynski, D. and Ahrends, R. and Mossmann, D. and Sickmann, A. and Zahedi, R.P. and Meisinger, C.
    Nature Communications 8 (2017)
    The mitochondrial proteome comprises ~1000 (yeast)-1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix. © 2017 The Author(s).
    view abstract10.1038/s41467-017-00359-0
  • LILY-lipidome isotope labeling of yeast:: In vivo synthesis of 13C labeled reference lipids for quantification by mass spectrometry
    Rampler, E. and Coman, C. and Hermann, G. and Sickmann, A. and Ahrends, R. and Koellensperger, G.
    Analyst 142 (2017)
    Quantification is an essential task in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity and their dynamic range of the lipidome. In this work, we introduce lipidome isotope labeling of yeast (LILY) in order to produce (non-radioactive) isotopically labeled eukaryotic lipid standards in yeast for normalization and quantification in mass spectrometric assays. More specifically, LILY is a fast and efficient in vivo labeling strategy in Pichia pastoris for the production of 13C labeled lipid library further paving the way to comprehensive compound-specific internal standardization in quantitative mass spectrometry based assays. More than 200 lipid species (from PA, PC, PE, PG, PI, PS, LysoGP, CL, DAG, TAG, DMPE, Cer, HexCer, IPC, MIPC) were obtained from yeast extracts with an excellent 13C enrichment &gt;99.5%, as determined by complementary high resolution mass spectrometry based shotgun and high resolution LC-MS/MS analysis. In a first proof of principle study we tested the relative and absolute quantification capabilities of the 13C enriched lipids obtained by LILY using a parallel reaction monitoring based LC-MS approach. In relative quantification it could be shown that compound specific internal standardization was essential for the accuracy extending the linear dynamic range to four orders of magnitude. Excellent analytical figures of merit were observed for absolute quantification for a selected panel of 5 investigated glycerophospholipids (e.g. LOQs around 5 fmol absolute; typical concentrations ranging between 1 to 10 nmol per 108 yeast cell starting material; RSDs &lt;10% (N = 4)). © The Royal Society of Chemistry 2017.
    view abstract10.1039/c7an00107j
  • Multi-OMICS: a critical technical perspective on integrative lipidomics approaches
    Kopczynski, D. and Coman, C. and Zahedi, R.P. and Lorenz, K. and Sickmann, A. and Ahrends, R.
    Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1862 (2017)
    During the past decades, high-throughput approaches for analyzing different molecular classes such as nucleic acids, proteins, metabolites, and lipids have grown rapidly. These approaches became powerful tools for getting a fundamental understanding of biological systems. Considering each approach and its results separately, relations and causal connections between these classes have no chance to be revealed, since only separate molecular snapshots are provided. Only a combined approach, not fully established yet, with the integration of the corresponding data, might yield a comprehensive and complete understanding of biological processes, such as crosstalk and interactions in signaling pathways. Taking two or more omics-methods into consideration for analysis is referred to as a multi-omics approach, which is gradually evolving. In this critical note, we briefly discuss the relevance, challenges, current state, and potential of data integration from multi-omics approaches, with a special focus on lipidomics analysis, listing the advantages and gaps in this field. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein. © 2017 Elsevier B.V.
    view abstract10.1016/j.bbalip.2017.02.003
  • PeptideMapper: Efficient and versatile amino acid sequence and tag mapping
    Kopczynski, D. and Barsnes, H. and Njølstad, P.R. and Sickmann, A. and Vaudel, M. and Ahrends, R.
    Bioinformatics 33 (2017)
    The mapping of amino acid sequences is an essential task in bioinformatics. Notably, the mapping of peptide sequences on a proteome is required for the post-processing of proteomics results. However, this step can quickly become a bottleneck when working with extensive numbers of peptides or large protein sequence databases. Here, we present PeptideMapper, a novel amino acid sequence mapper for both peptide sequences and de novo sequencing identification results. By taking advantage of the latest advances in pattern matching, PeptideMapper achieves unprecedented performance (i.e. up to 1000 faster than state-of-the-art software) in terms of memory footprint and execution speed, with regards to both the indexing and the querying of protein sequence databases. Availability and Implementation: PeptideMapper is implemented in the open source Java CompOmics framework under the permissive Apache 2.0 license © 2017 The Author.
    view abstract10.1093/bioinformatics/btx122
  • PeptideTracker: A knowledge base for collecting and storing information on protein concentrations in biological tissues
    Mohammed, Y. and Bhowmick, P. and Smith, D.S. and Domanski, D. and Jackson, A.M. and Michaud, S.A. and Malchow, S. and Percy, A.J. and Chambers, A.G. and Palmer, A. and Zhang, S. and Sickmann, A. and Borchers, C.H.
    Proteomics 17 (2017)
    view abstract10.1002/pmic.201600210
  • Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin
    Gonczarowska-Jorge, H. and Loroch, S. and Dell'Aica, M. and Sickmann, A. and Roos, A. and Zahedi, R.P.
    Analytical Chemistry 89 (2017)
    Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, nonunique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as Glu-C, Asp-N, Lys-N, Arg-C, LysargiNase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1-5 min. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8363 vs 6807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14:628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either Asp-N, Arg-C, Glu-C, Lys-C, or Lys-N. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsin - despite broad specificity and fast digestion that may increase technical variance. © 2017 American Chemical Society.
    view abstract10.1021/acs.analchem.7b02395
  • Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition
    Beck, F. and Geiger, J. and Gambaryan, S. and Solari, F.A. and Dell'aica, M. and Loroch, S. and Mattheij, N.J. and Mindukshev, I. and Pötz, O. and Jurk, K. and Burkhart, J.M. and Fufezan, C. and Heemskerk, J.W.M. and Walter, U. and Zahedi, R.P. and Sickmann, A.
    Blood 129 (2017)
    Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin aIIbb3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determinedhow Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPaseactivating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. © 2017 by The American Society of Hematology.
    view abstract10.1182/blood-2016-05-714048
  • The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier
    Schell, C. and Rogg, M. and Suhm, M. and Helmstädter, M. and Sellung, D. and Yasuda-Yamahara, M. and Kretz, O. and Küttner, V. and Suleiman, H. and Kollipara, L. and Zahedi, R.P. and Sickmann, A. and Eimer, S. and Shaw, A.S. and Kramer-Zucker, A. and Hirano-Kobayashi, M. and Abe, T. and Aizawa, S. and Grahammer, F. and Hartleben, B. and Dengjel, J. and Huber, T.B.
    Proceedings of the National Academy of Sciences of the United States of America 114 (2017)
    Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrixdependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.
    view abstract10.1073/pnas.1617004114
  • The mTOR and PP2A Pathways Regulate PHD2 Phosphorylation to Fine-Tune HIF1α Levels and Colorectal Cancer Cell Survival under Hypoxia
    Di Conza, G. and Trusso Cafarello, S. and Loroch, S. and Mennerich, D. and Deschoemaeker, S. and Di Matteo, M. and Ehling, M. and Gevaert, K. and Prenen, H. and Zahedi, R.P. and Sickmann, A. and Kietzmann, T. and Moretti, F. and Mazzone, M.
    Cell Reports 18 (2017)
    Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth. © 2017 The Author(s)
    view abstract10.1016/j.celrep.2017.01.051
  • The proteome of baker's yeast mitochondria
    Gonczarowska-Jorge, H. and Zahedi, R.P. and Sickmann, A.
    Mitochondrion 33 (2017)
    In the past decade mass spectrometry-based proteomics has greatly contributed to shaping our knowledge about . Saccharomyces cerevisiae mitochondria, from the initial identification of novel essential components in purified protein complexes, to the actual characterization of the mitochondrial proteome, the specific analysis of mitochondrial subcompartment proteomes, and the study of regulatory mechanisms that govern mitochondrial homeostasis. Here, we provide an overview of relevant mitochondrial proteome studies and furthermore discuss future possibilities how proteomics will further improve our existing understanding of mitochondria. Although mitochondria were the first organelles that have been intensively studied using proteomics, indeed the recent progress and development of more powerful and sensitive methods, instrumentation and data analysis strategies indicate that we are only beginning to exploit the full potential of mitochondrial proteomics and its possibilities to decipher cell biology. Beside mere (quantitative) inventory under different conditions, this will for instance include studying the role of multiple post-translational modifications in mitochondrial homeostasis as well as the system-wide mapping of protein-protein complexes and protein-lipid interactions. © 2016.
    view abstract10.1016/j.mito.2016.08.007
  • Tracking Effects of SIL1 Increase: Taking a Closer Look Beyond the Consequences of Elevated Expression Level
    Labisch, T. and Buchkremer, S. and Phan, V. and Kollipara, L. and Gatz, C. and Lentz, C. and Nolte, K. and Vervoorts, J. and Coraspe, J.A.G. and Sickmann, A. and Carr, S. and Zahedi, R.P. and Weis, J. and Roos, A.
    Molecular Neurobiology (2017)
    SIL1 acts as a co-chaperone for the major ER-resident chaperone BiP and thus plays a role in many BiP-dependent cellular functions such as protein-folding control and unfolded protein response. Whereas the increase of BiP upon cellular stress conditions is a well-known phenomenon, elevation of SIL1 under stress conditions was thus far solely studied in yeast, and different studies indicated an adverse effect of SIL1 increase. This is seemingly in contrast with the beneficial effect of SIL1 increase in surviving neurons in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer’s disease. Here, we addressed these controversial findings. Applying cell biological, morphological and biochemical methods, we demonstrated that SIL1 increases in various mammalian cells and neuronal tissues upon cellular stress. Investigation of heterozygous SIL1 mutant cells and tissues supported this finding. Moreover, SIL1 protein was found to be stabilized during ER stress. Increased SIL1 initiates ER stress in a concentration-dependent manner which agrees with the described adverse SIL1 effect. However, our results also suggest that protective levels are achieved by the secretion of excessive SIL1 and GRP170 and that moderately increased SIL1 also ameliorates cellular fitness under stress conditions. Our immunoprecipitation results indicate that SIL1 might act in a BiP-independent manner. Proteomic studies showed that SIL1 elevation alters the expression of proteins including crucial players in neurodegeneration, especially in Alzheimer’s disease. This finding agrees with our observation of increased SIL1 immunoreactivity in surviving neurons of Alzheimer’s disease autopsy cases and supports the assumption that SIL1 plays a protective role in neurodegenerative disorders. © 2017 Springer Science+Business Media New York
    view abstract10.1007/s12035-017-0494-6
  • A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii
    Lefebvre-Legendre, L. and Reifschneider, O. and Kollipara, L. and Sickmann, A. and Wolters, D. and Kück, U. and Goldschmidt-Clermont, M.
    Plant Journal 85 (2016)
    Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity. © 2015 The Authors The Plant Journal.
    view abstract10.1111/tpj.13089
  • A Ribonucleoprotein supercomplex involved in trans-splicing of organelle group II Introns
    Reifschneider, O. and Marx, C. and Jacobs, J. and Kollipara, L. and Sickmann, A. and Wolters, D. and Kück, U.
    Journal of Biological Chemistry 291 (2016)
    In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaAi2, splice in trans, and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatographymass spectrometry (LC-MS), size exclusion chromatography, and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein apparatus participating in psaA mRNA splicing. This supercomplex contains two subcomplexes (I and II) that are responsible for trans-splicing of either psaA-i1 or psaA-i2. We further demonstrate that both subcomplexes are associated with intron RNA, which is a prerequisite for the correct assembly of subcomplex I. This study contributes further to our view of how the eukaryotic nuclear spliceosome evolved after bacterial endosymbiosis through fragmentation of self-splicing group II introns into a dynamic, protein-rich RNP machinery.
    view abstract10.1074/jbc.M116.750570
  • Annotated Gene and Proteome Data Support Recognition of Interconnections Between the Results of Different Experiments in Space Research
    Bauer, J. and Wehland, M. and Pietsch, J. and Sickmann, A. and Weber, G. and Grimm, D.
    Microgravity Science and Technology 28 (2016)
    In a series of studies, human thyroid and endothelial cells exposed to real or simulated microgravity were analyzed in terms of changes in gene expression patterns or protein content. Due to the limitation of available cells in many space research experiments, comparative and control experiments had to be done in a serial manner. Therefore, detected genes or proteins were annotated with gene names and SwissProt numbers, in order to allow searches for interconnections between results obtained in different experiments by different methods. A crosscheck of several studies on the behavior of cytoskeletal genes and proteins suggested that clusters of cytoskeletal components change differently under the influence of microgravity and/or vibration in different cell types. The result that LOX and ISG15 gene expression were clearly altered during the Shenzhou-8 spaceflight mission could be estimated by comparison with the results of other experiments. The more than 100-fold down-regulation of LOX supports our hypothesis that the amount and stability of extracellular matrix have a great influence on the formation of three-dimensional aggregates under microgravity. The approximately 40-fold up-regulation of ISG15 cannot yet be explained in detail, but strongly suggests that ISGylation, an alternative form of posttranslational modification, plays a role in longterm cultures. © 2015, Springer Science+Business Media Dordrecht.
    view abstract10.1007/s12217-015-9451-z
  • Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome
    Ciregia, F. and Kollipara, L. and Giusti, L. and Zahedi, R. P. and Giacomelli, C. and Mazzoni, M. R. and Giannaccini, G. and Scarpellini, P. and Urbani, A. and Sickmann, A. and Lucacchini, A. and Bazzichi, L.
    Translational Psychiatry 6 (2016)
    Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n = 45 CFS; n = 45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.
    view abstract10.1038/tp.2016.184
  • Combined quantification of the global proteome, phosphoproteome, and proteolytic cleavage to characterize altered platelet functions in the human scott syndrome
    Solari, F.A. and Mattheij, N.J.A. and Burkhart, J.M. and Swieringa, F. and Collins, P.W. and Cosemans, J.M.E.M. and Sickmann, A. and Heemskerk, J.W.M. and Zahedi, R.P.
    Molecular and Cellular Proteomics 15 (2016)
    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multipronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspaseregulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
    view abstract10.1074/mcp.M116.060368
  • Functional dissection of an alternatively spliced herpesvirus gene by splice site mutagenesis
    Schommartz, T. and Loroch, S. and Alawi, M. and Grundhoff, A. and Sickmann, A. and Brune, W.
    Journal of Virology 90 (2016)
    Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. © 2016, American Society for Microbiology. All Rights Reserved.
    view abstract10.1128/JVI.02987-15
  • Global profiling of protein complexes: current approaches and their perspective in biomedical research
    Rudashevskaya, E.L. and Sickmann, A. and Markoutsa, S.
    Expert Review of Proteomics 13 (2016)
    Introduction: Despite the rapid evolution of proteomic methods, protein interactions and their participation in protein complexes – an important aspect of their function – has rarely been investigated on the proteome-wide level. Disease states, such as muscular dystrophy or viral infection, are induced by interference in protein-protein interactions within complexes. The purpose of this review is to describe the current methods for global complexome analysis and to critically discuss the challenges and opportunities for the application of these methods in biomedical research. Areas covered: We discuss advancements in experimental techniques and computational tools that facilitate profiling of the complexome. The main focus is on the separation of native protein complexes via size exclusion chromatography and gel electrophoresis, which has recently been combined with quantitative mass spectrometry, for a global protein-complex profiling. The development of this approach has been supported by advanced bioinformatics strategies and fast and sensitive mass spectrometers that have allowed the analysis of whole cell lysates. The application of this technique to biomedical research is assessed, and future directions are anticipated. Expert commentary: The methodology is quite new, and has already shown great potential when combined with complementary methods for detection of protein complexes. © 2016 Informa UK Limited, trading as Taylor & Francis Group.
    view abstract10.1080/14789450.2016.1233064
  • Simultaneous metabolite, protein, lipid extraction (SIMPLEX): A combinatorial multimolecular omics approach for systems biology
    Coman, C. and Solari, F.A. and Hentschel, A. and Sickmann, A. and Zahedi, R.P. and Ahrends, R.
    Molecular and Cellular Proteomics 15 (2016)
    Interconnected molecular networks are at the heart of signaling pathways that mediate adaptive plasticity of eukaryotic cells. To gain deeper insights into the underlying molecular mechanisms, a comprehensive and representative analysis demands a deep and parallel coverage of a broad spectrum of molecular species. Therefore, we introduce a simultaneous metabolite, protein, lipid extraction (SIMPLEX) procedure, a novel strategy for the quantitative investigation of lipids, metabolites, and proteins. Compared with unimolecular workflows, SIMPLEX offers a fundamental turn in study design since multiple molecular classes can be accessed in parallel from one sample with equal efficiency and reproducibility. Application of this method in mass-spectrometry-based workflows allowed the simultaneous quantification of 360 lipids, 75 metabolites, and 3327 proteins from 106 cells. The versatility of this method is shown in a model system for adipogenesis-peroxisomal proliferator-activated receptor gamma (PPARG) signaling in mesenchymal stem cells-where we utilized SIMPLEX to explore cross-talk within and between all three molecular classes and identified novel potential molecular entry points for interventions, indicating that SIMPLEX provides a superior strategy compared with conventional workflows. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
    view abstract10.1074/mcp.M115.053702
  • Two birds with one stone: Parallel quantifi cation of proteome and phosphoproteome using iTRAQ
    Solari, F.A. and Kollipara, L. and Sickmann, A. and Zahedi, R.P.
    Methods in Molecular Biology 1394 (2016)
    Altered and abnormal levels of proteins and their phosphorylation states are associated with many disorders. Detection and quantification of such perturbations may provide a better understanding of pathological conditions and help finding candidates for treatment or biomarkers. Over the years, isobaric mass tags for relative quantification of proteins and protein phosphorylation by mass spectrometry have become increasingly popular. One of the most commonly used isobaric chemical tags is iTRAQ (isobaric tag for relative and absolute quantitation). In a typical iTRAQ-8plex experiment, a multiplexed sample amounts for up to 800 μg of peptides. Using state-of-the-art LC-MS approaches, only a fraction (~5 %) of such a sample is required to generate comprehensive quantitative data on the global proteome level, so that the bulk of the sample can be simultaneously used for quantitative phosphoproteomics. Here, we provide a simple and straightforward protocol to perform quantitative analyses of both proteome and phosphoproteome from the same sample using iTRAQ. © Springer Science+Business Media New York 2016.
    view abstract10.1007/978-1-4939-3341-9_3
  • “Watching the Detectives” report of the general assembly of the EU project DETECTIVE Brussels, 24–25 November 2015
    Fernando, R.N. and Chaudhari, U. and Escher, S.E. and Hengstler, J.G. and Hescheler, J. and Jennings, P. and Keun, H.C. and Kleinjans, J.C.S. and Kolde, R. and Kollipara, L. and Kopp-Schneider, A. and Limonciel, A. and Nemade, H. and Nguemo, F. and Peterson, H. and Prieto, P. and Rodrigues, R.M. and Sachinidis, A. and Schäfer, C. and Sickmann, A. and Spitkovsky, D. and Stöber, R. and van Breda, S.G.J. and van de Water, B. and Vivier, M. and Zahedi, R.P. and Vinken, M. and Rogiers, V.
    Archives of Toxicology 90 (2016)
    SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and “-omics” technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich “-omics” databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project. © 2016, Springer-Verlag Berlin Heidelberg.
    view abstract10.1007/s00204-016-1719-6
  • Conjugation of ciprofloxacin with poly(2-oxazoline)s and polyethylene glycol via end groups
    Schmidt, M. and Harmuth, S. and Barth, E.R. and Wurm, E. and Fobbe, R. and Sickmann, A. and Krumm, C. and Tiller, J.C.
    Bioconjugate Chemistry 26 (2015)
    The antibiotic ciprofloxacin (CIP) was covalently attached to the chain end of poly(2-methyloxazoline) (PMOx), poly(2-ethyloxazoline) (PEtOx), and polyethylene glycol (PEG), and the antimicrobial activity of these conjugates was tested for Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, and Kleisella pneumoniae. Chemical structures of the conjugates were proven by 1H NMR and electron spray ionization mass spectrometry. The direct coupling of PMOx and CIP resulted in low antimicrobial activity. The coupling via a spacer afforded molecular weight dependent activity with a molar minimal inhibitory concentration that is even higher than that of the pristine CIP. The antimicrobial activity of the conjugates increases in the order of PMOx < PEtOx < PEG. Conjugation of CIP and a quaternary ammonium compound via PMOx did not result in higher activity, indicating no satellite group or synergistic effect of the different biocidal end groups. © 2015 American Chemical Society.
    view abstract10.1021/acs.bioconjchem.5b00393
  • Current strategies and findings in clinically relevant post-translational modification-specific proteomics
    Pagel, O. and Loroch, S. and Sickmann, A. and Zahedi, R.P.
    Expert Review of Proteomics 12 (2015)
    Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research. © Informa Uk, Ltd.
    view abstract10.1586/14789450.2015.1042867
  • Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III
    Björkqvist, J. and De Maat, S. and Lewandrowski, U. and Di Gennaro, A. and Oschatz, C. and Schönig, K. and Nöthen, M.M. and Drouet, C. and Braley, H. and Nolte, M.W. and Sickmann, A. and Panousis, C. and Maas, C. and Renné, T.
    Journal of Clinical Investigation 125 (2015)
    Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes. © 2015, American Society for Clinical Investigation. All rights reserved.
    view abstract10.1172/JCI77139
  • Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography
    Loroch, S. and Zahedi, R.P. and Sickmann, A.
    Analytical Chemistry 87 (2015)
    In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 107 down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti4+, our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research. © 2014 American Chemical Society.
    view abstract10.1021/ac502708m
  • Identification of novel mechanistic biomarkers of nephrotoxicity by proteomics and phosphoproteomics
    Limonciel, A. and Kollipara, L. and Zahedi, R. and Sickmann, A. and Jennings, P.
    Toxicology Letters 238 (2015)
    view abstract10.1016/j.toxlet.2015.08.685
  • Identification of proteins involved in inhibition of spheroid formation under microgravity
    Riwaldt, S. and Pietsch, J. and Sickmann, A. and Bauer, J. and Braun, M. and Segerer, J. and Schwarzwälder, A. and Aleshcheva, G. and Corydon, T.J. and Infanger, M. and Grimm, D.
    Proteomics 15 (2015)
    Many types of cells transit in vitro from a two- to a three-dimensional growth, when they are exposed to microgravity. The underlying mechanisms are not yet understood. Hence, we investigated the impact of microgravity on protein content and growth behavior. For this purpose, the human thyroid cancer cells FTC-133 were seeded either in recently developed cell containers that can endure enhanced physical forces and perform media changes and cell harvesting automatically or in T-25 culture flasks. All cells were cultured for five days at 1g. Afterwards, a part of the cell containers were flown to the International Space Station, while another part was kept on the ground. T-25 flasks were mounted on and next to a Random Positioning Machine. The cells were cultured for 12 days under the various conditions, before they were fixed with RNAlater. All fixed cultures showed monolayers, but three-dimensional aggregates were not detected. In a subsequent protein analysis, 180 proteins were identified by mass spectrometry. These proteins did not indicate significant differences between cells exposed to microgravity and their 1g controls. However, they suggest that an enhanced production of proteins related to the extracellular matrix could detain the cells from spheroid formation, while profilin-1 is phosphorylated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201500067
  • Mass spectrometry-based proteomics for relative protein quantification and biomarker identification in primary human hepatocytes
    Dietz, L. and Sickmann, A.
    Methods in Molecular Biology 1250 (2015)
    Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The twodimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results. © Springer Science+Business Media New York 2015.
    view abstract10.1007/978-1-4939-2074-7_18
  • Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research
    Loroch, S. and Schommartz, T. and Brune, W. and Zahedi, R.P. and Sickmann, A.
    Biochimica et Biophysica Acta - Proteins and Proteomics 1854 (2015)
    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ∼ 75% more proteins and even ∼ 134% more peptides compared to the 2D strategy. Additionally, we could quantify ∼ 50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. © 2015 Published by Elsevier B.V.
    view abstract10.1016/j.bbapap.2015.01.006
  • PeptideShaker enables reanalysis of MS-derived proteomics data sets: To the editor
    Vaudel, M. and Burkhart, J.M. and Zahedi, R.P. and Oveland, E. and Berven, F.S. and Sickmann, A. and Martens, L. and Barsnes, H.
    Nature Biotechnology 33 (2015)
    view abstract10.1038/nbt.3109
  • Why phosphoproteomics is still a challenge
    Solari, F.A. and Dell'Aica, M. and Sickmann, A. and Zahedi, R.P.
    Molecular BioSystems 11 (2015)
    Despite continuous improvements phosphoproteomics still faces challenges that are often neglected, e.g. partially poor recovery of phosphopeptide enrichment, assessment of phosphorylation stoichiometry, label-free quantification, poor behavior during chromatography, and general limitations of peptide-centric proteomics. Here we critically discuss current limitations that need consideration in both qualitative and quantitative studies. © The Royal Society of Chemistry 2015.
    view abstract10.1039/c5mb00024f
  • Amyloid-β Peptide Induces Mitochondrial Dysfunction by Inhibition of Preprotein Maturation
    Mossmann, D. and Vögtle, F.-N. and Taskin, A. and Teixeira, P. and Ring, J. and Burkhart, JuliaM. and Burger, N. and Pinho, C. and Tadic, J. and Loreth, D. and Graff, C. and Metzger, F. and Sickmann, A. and Kretz, O. and Wiedemann, N. and Zahedi, RenP. and Madeo, F. and Glaser, E. and Meisinger, C.
    Cell Metabolism 20 (2014)
    Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aβ). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aβ inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aβ peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease. © 2014 Elsevier Inc. All rights reserved.
    view abstract10.1016/j.cmet.2014.07.024
  • Antimicrobial poly(2-methyloxazoline)s with bioswitchable activity through satellite group modification
    Krumm, C. and Harmuth, S. and Hijazi, M. and Neugebauer, B. and Kampmann, A.-L. and Geltenpoth, H. and Sickmann, A. and Tiller, J.C.
    Angewandte Chemie - International Edition 53 (2014)
    Biocides are widely used for preventing the spread of microbial infections and fouling of materials. Since their use can build up microbial resistance and cause unpredictable long-term environmental problems, new biocidal agents are required. In this study, we demonstrate a concept in which an antimicrobial polymer is deactivated by the cleavage of a single group. Following the satellite group approach, a biocidal quaternary ammonium group was linked through a poly(2-methyloxazoline) to an ester satellite group. The polymer with an octyl-3-propionoate satellite group shows very good antimicrobial activity against Gram-positive bacterial strains. The biocidal polymer was also found to have low hemotoxicity, resulting in a high HC50/MIC value of 120 for S. aureus. Cleaving the ester satellite group resulted in a 30-fold decrease in antimicrobial activity, proving the concept valid. The satellite group could also be cleaved by lipase showing that the antimicrobial activity of the new biocidal polymers is indeed bioswitchable. Biocides are widely used for preventing the spread of microbial infections and the fouling of materials. Since their application can build up microbial resistance and cause unpredictable long-term environmental problems, new biocidal agents are required. In a novel approach an antimicrobial polymer is deactivated by hydrolysis of an ester group through the action of a lipase. The crucial feature is the mutual interaction of the two endgroups of the polymer. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/anie.201311150
  • Catch me if you can: Challenges and applications of cross-linking approaches
    Tinnefeld, V. and Sickmann, A. and Ahrends, R.
    European Journal of Mass Spectrometry 20 (2014)
    Biomolecular complexes are the groundwork of life and the basis for cell signaling, energy transfer, motion, stability and cellular metabolism. Understanding the underlying complex interactions on the molecular level is an essential step to obtain a comprehensive insight into cellular and systems biology. For the investigation of molecular interactions, various methods, including Förster resonance energy transfer, nuclear magnetic resonance spectroscopy, X-ray crystallography and yeast two-hybrid screening, can be utilized. Nevertheless, the most reliable approach for structural proteomics and the identification of novel protein-binding partners is chemical cross-linking. The rationale is that upon forming a covalent bond between a protein and its interaction partner (protein, lipid, RNA/DNA, carbohydrate) the native complex state is "frozen" and accessible for detailed mass spectrometric analysis. In this review we provide a synopsis on crosslinker design, chemistry, pitfalls, limitations and novel applications in the field, and feature an overview of current software applications. © 2014 IM Publications LLP.
    view abstract10.1255/ejms.1259
  • Introduction to opportunities and pitfalls in functional mass spectrometry based proteomics
    Vaudel, M. and Sickmann, A. and Martens, L.
    Biochimica et Biophysica Acta - Proteins and Proteomics 1844 (2014)
    With the advent of mass spectrometry based proteomics, the identification of thousands of proteins has become commonplace in biology nowadays. Increasingly, efforts have also been invested toward the detection and localization of posttranslational modifications. It is furthermore common practice to quantify the identified entities, a task supported by a panel of different methods. Finally, the results can also be enriched with functional knowledge gained on the proteins, detecting for instance differentially expressed gene ontology terms or biological pathways. In this study, we review the resources, methods and tools available for the researcher to achieve such a quantitative functional analysis. These include statistics for the post-processing of identification and quantification results, online resources and public repositories. With a focus on free but user-friendly software, preferably also open-source, we provide a list of tools designed to help the researcher manage the vast amount of data generated. We also indicate where such applications currently remain lacking. Moreover, we stress the eventual pitfalls of every step of such studies. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan. © 2013 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.bbapap.2013.06.019
  • Proteomic differences between microvascular endothelial cells and the EA.hy926 cell line forming three-dimensional structures
    Ma, X. and Sickmann, A. and Pietsch, J. and Wildgruber, R. and Weber, G. and Infanger, M. and Bauer, J. and Grimm, D.
    Proteomics 14 (2014)
    Proteomic changes of two types of human endothelial cells (ECs) were determined and compared to morphological alterations occurring during the scaffold-free in vitro formation of 3D structures resembling vascular intimas. The EA.hy926 cell line and human microvascular ECs (HMVECs) were cultured on a random positioning machine or static on ground (normal gravity) for 5 and 7 days, before their morphology was examined and their protein content was analysed by MS after free-flow electrophoretic separation. A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVEC forming 3D structures faster than the EA.hy926 cells. Five hundred and eighty-four of these kinds of proteins were present in both types of cells. They included a number of metabolic enzymes, of structure-related and stress proteins. Comparing proteins of EA.hy926 cells growing either adherently on ground or in 3D aggregates on the random positioning machine revealed that ribosomal proteins were enhanced, while tubes are formed and various components of 26S proteasomes remained prevalent in static normal gravity control cells only. The fast developing tube-like 3D structures of HMVEC suggested a transient augmentation of ribosomal proteins during the 3D assembling of ECs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201300453
  • Proteomics - moving from inventory to personalized medicine?
    Zahedi, R.P. and Ueffing, M. and Sickmann, A.
    Proteomics 14 (2014)
    view abstract10.1002/pmic.201470133
  • The protein import machinery of mitochondria - A regulatory hub in metabolism, stress, and disease
    Harbauer, A.B. and Zahedi, R.P. and Sickmann, A. and Pfanner, N. and Meisinger, C.
    Cell Metabolism 19 (2014)
    Mitochondria fulfill central functions in bioenergetics, metabolism, and apoptosis. They import more than 1,000 different proteins from the cytosol. It had been assumed that the protein import machinery is constitutively active and not subject to detailed regulation. However, recent studies indicate that mitochondrial protein import is regulated at multiple levels connected to cellular metabolism, signaling, stress, and pathogenesis of diseases. Here, we discuss the molecular mechanisms of import regulation and their implications for mitochondrial homeostasis. The protein import activity can function as a sensor of mitochondrial fitness and provides a direct means of regulating biogenesis, composition, and turnover of the organelle. © 2014 Elsevier Inc.
    view abstract10.1016/j.cmet.2014.01.010
  • Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways
    Beck, F. and Geiger, J. and Gambaryan, S. and Veit, J. and Vaudel, M. and Nollau, P. and Kohlbacher, O. and Martens, L. and Walter, U. and Sickmann, A. and Zahedi, R.P.
    Blood 123 (2014)
    One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-signaling cascade and inhibits virtually all platelet-activating key mechanisms. Using quantitative mass spectrometry, we analyzed time-resolved phosphorylation patterns in human platelets after treatment with iloprost, a stable prostacyclin analog, for 0, 10, 30, and 60 seconds to characterize key mediators of platelet inhibition and activation in 3 independent biological replicates. We quantified over 2700 different phosphorylated peptides of which 360 were significantly regulated upon stimulation. This comprehensive and time-resolved analysis indicates that platelet inhibition is a multipronged process involving different kinases and phosphatases aswell asmany previously unanticipated proteins and pathways. © 2014 by The American Society of Hematology.
    view abstract10.1182/blood-2013-07-512384
  • What can proteomics tell us about platelets?
    Burkhart, J.M. and Gambaryan, S. and Watson, S.P. and Jurk, K. and Walter, U. and Sickmann, A. and Heemskerk, J.W.M. and Zahedi, R.P.
    Circulation Research 114 (2014)
    More than 130 years ago, it was recognized that platelets are key mediators of hemostasis. Nowadays, it is established that platelets participate in additional physiological processes and contribute to the genesis and progression of cardiovascular diseases. Recent data indicate that the platelet proteome, defined as the complete set of expressed proteins, comprises >5000 proteins and is highly similar between different healthy individuals. Owing to their anucleate nature, platelets have limited protein synthesis. By implication, in patients experiencing platelet disorders, platelet (dys)function is almost completely attributable to alterations in protein expression and dynamic differences in post-translational modifications. Modern platelet proteomics approaches can reveal (1) quantitative changes in the abundance of thousands of proteins, (2) post-translational modifications, (3) protein-protein interactions, and (4) protein localization, while requiring only small blood donations in the range of a few milliliters. Consequently, platelet proteomics will represent an invaluable tool for characterizing the fundamental processes that affect platelet homeostasis and thus determine the roles of platelets in health and disease. In this article we provide a critical overview on the achievements, the current possibilities, and the future perspectives of platelet proteomics to study patients experiencing cardiovascular, inflammatory, and bleeding disorders.
    view abstract10.1161/CIRCRESAHA.114.301598
  • Adenylylation, MS, and proteomics-Introducing a "new" modification to bottom-up proteomics
    Hansen, T. and Albers, M. and Hedberg, C. and Sickmann, A.
    Proteomics 13 (2013)
    Although the addition of a 5′-adenosine phosphodiester group to proteins, called adenylylation, has been known for decades, the possibility that adenylylation could be a molecular switch in cellular signaling pathways has emerged recently. The distinct mass shift upon adenylation of threonine or tyrosine residues renders it a good target for MS detection and identification; however, the fragmentation of adenylylated peptides derived from proteolytic digestion of adenylylated proteins has not yet been systematically investigated. Here, we demonstrate that adenylylated peptides show loss of parts of the adenosine monophosphate (AMP) upon different fragmentation techniques. As expected, causing the least fragmentation of the AMP group, electron transfer dissociation yields less complicated spectra. In contrast, CID and higher energy collision (HCD) fragmentation caused AMP to fragment, generating characteristic ions that could be utilized in the specific identification of adenylylated peptides. The characteristic ions and losses upon CID and higher energy collision fragmentation from the AMP group turned out to be highly dependent on which amino acid was adenylylated, with different reporter ions for adenylylated threonine and tyrosine. We also investigated how adenylylation is best incorporated into search engines, exemplified by Mascot and showed that it is possible to identify adenylylation by search engines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201200344
  • An alternative NFAT-activation pathway mediated by IL-7 is critical for early thymocyte development
    Patra, A.K. and Avots, A. and Zahedi, R.P. and Schüler, T. and Sickmann, A. and Bommhardt, U. and Serfling, E.
    Nature Immunology 14 (2013)
    Interleukin 7 (IL-7) has a critical role in the development of early CD4-CD8-double-negative (DN) thymocytes. Although the transcription factor STAT5 is an important component of IL-7 signaling, differences in the phenotypes of mice deficient in STAT5, IL-7, IL-7 receptor alpha (IL-7rα) or the kinase Jak3 suggest the existence of STAT5-independent IL-7 signaling. Here we found that IL-7-Jak3 signals activated the transcription factor NFATc1 in DN thymocytes by phosphorylating Tyr371 in the regulatory region of NFATc1. This NFAT-activation pathway was critical for the survival and development of DN thymocytes, as deficiency in NFATc1 blocked thymocyte development at the DN1 stage, leading to T cell lymphopenia. In addition, our results demonstrated a cooperative function for NFATc1 and STAT5 in guiding thymocyte development in response to IL-7 signals. © 2013 Nature America, Inc. All rights reserved.
    view abstract10.1038/ni.2507
  • Analysis of Post-translational Modifications
    Zahedi, R. and Sickmann, A.
    Proteomics 13 (2013)
    view abstract10.1002/pmic.201370054
  • Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response
    Stahl, S. and Burkhart, J.M. and Hinte, F. and Tirosh, B. and Mohr, H. and Zahedi, R.P. and Sickmann, A. and Ruzsics, Z. and Budt, M. and Brune, W.
    PLoS Pathogens 9 (2013)
    During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR. © 2013 Stahl et al.
    view abstract10.1371/journal.ppat.1003544
  • Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling
    Schweigel, H. and Geiger, J. and Beck, F. and Buhs, S. and Gerull, H. and Walter, U. and Sickmann, A. and Nollau, P.
    Proteomics 13 (2013)
    Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201200353
  • Developmental changes of the protein repertoire in the rat auditory brainstem: A comparative proteomics approach in the superior olivary complex and the inferior colliculus with DIGE and iTRAQ
    Kaltwaßer, B. and Schulenborg, T. and Beck, F. and Klotz, M. and Schäfer, K.-H. and Schmitt, M. and Sickmann, A. and Friauf, E.
    Journal of Proteomics 79 (2013)
    Protein profiles of developing neural circuits undergo manifold changes. The aim of this proteomic analysis was to quantify postnatal changes in two auditory brainstem areas in a comparative approach. Protein samples from the inferior colliculus (IC) and the superior olivary complex (SOC) were obtained from neonatal (P4) and young adult (P60) rats. The cytosolic fractions of both areas were examined by 2-D DIGE, and the plasma membrane-enriched fraction of the IC was analyzed via iTRAQ. iTRAQ showed a regulation in 34% of the quantified proteins. DIGE revealed 12% regulated spots in both the SOC and IC and, thus, numeric congruency. Although regulation in KEGG pathways displayed a similar pattern in both areas, only 13 of 71 regulated DIGE proteins were regulated in common, implying major area-specific differences. 89% of regulated glycolysis/gluconeogenesis and citrate cycle proteins were up-regulated in the SOC or IC, suggesting a higher energy demand in adulthood. Seventeen cytoskeleton proteins were regulated, consistent with complex morphological reorganization between P4 and P60. Fourteen were uniquely regulated in the SOC, providing further evidence for area-specific differences. Altogether, we provide the first elaborate catalog of proteins involved in auditory brainstem development, several of them possibly of particular developmental relevance. © 2012 Elsevier B.V.
    view abstract10.1016/j.jprot.2012.11.018
  • Interaction of proteins identified in human thyroid cells
    Pietsch, J. and Riwaldt, S. and Bauer, J. and Sickmann, A. and Weber, G. and Grosse, J. and Infanger, M. and Eilles, C. and Grimm, D.
    International Journal of Molecular Sciences 14 (2013)
    Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. © 2013 by the authors; licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ijms14011164
  • Interleukin-7-mediated NFAT activation sets the threshold for T cell development
    Patra, A. K. and Avots, A. and Zahedi, R. P. and Schuler, T. and Sickmann, A. and Bommhardt, U. and Serfling, E.
    Cytokine 63 (2013)
    view abstract10.1016/j.cyto.2013.06.199
  • Method and platform standardization in MRM-based quantitative plasma proteomics
    Percy, A.J. and Chambers, A.G. and Yang, J. and Jackson, A.M. and Domanski, D. and Burkhart, J. and Sickmann, A. and Borchers, C.H.
    Journal of Proteomics 95 (2013)
    There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. Biological significance: The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms.This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. © 2013 Elsevier B.V.
    view abstract10.1016/j.jprot.2013.07.026
  • Novel function assignment to a member of the essential HP1043 response regulator family of epsilon-proteobacteria
    Bauer, S. and Endres, M. and Lange, M. and Schmidt, T. and Schumbrutzki, C. and Sickmann, A. and Beier, D.
    Microbiology (United Kingdom) 159 (2013)
    HP1043 of Helicobacter pylori is an orphan response regulator (RR) with a highly degenerate receiver sequence incapable of phosphorylation, which is essential for cell viability. In contrast, the orthologous RR protein of Helicobacter pullorum, an enterohepatic Helicobacter species mainly isolated from poultry, harbours a consensus receiver sequence and is associated with a cognate histidine kinase (HK). Here, we show that this two-component system of H. pullorum, denoted HPMG439/HPMG440, is involved in the control of nitrogen metabolism by regulating the expression of glutamate dehydrogenase, an AmtB ammonium transporter and a PII protein. However, the role of the RR HPMG439 is not restricted to nitrogen regulation since, in contrast with the HK HPMG440, HPMG439 is essential for growth of H. pullorum under nutrient-rich conditions. © 2013 SGM.
    view abstract10.1099/mic.0.066548-0
  • Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55
    Venne, A.S. and Vögtle, F.-N. and Meisinger, C. and Sickmann, A. and Zahedi, R.P.
    Journal of Proteome Research 12 (2013)
    We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 μg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities. © 2013 American Chemical Society.
    view abstract10.1021/pr400435d
  • Nuclear import of LASP-1 is regulated by phosphorylation and dynamic protein-protein interactions
    Mihlan, S. and Reiß, C. and Thalheimer, P. and Herterich, S. and Gaetzner, S. and Kremerskothen, J. and Pavenstädt, H.J. and Lewandrowski, U. and Sickmann, A. and Butt, E.
    Oncogene 32 (2013)
    LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression. © 2013 Macmillan Publishers Limited All rights reserved.
    view abstract10.1038/onc.2012.216
  • Phosphoproteomics-More than meets the eye
    Loroch, S. and Dickhut, C. and Zahedi, R.P. and Sickmann, A.
    Electrophoresis 34 (2013)
    PTMs enable cells to adapt to internal and external stimuli in the milliseconds to seconds time regime. Protein phosphorylation is probably the most important of these modifications as it affects protein structure and interactions, critically influencing the life cycle of a cell. In the last 15 years, new insights into phosphorylation have been provided by highly sensitive MS-based approaches combined with specific phosphopeptide enrichment strategies. Although so far research has mainly focused on the discovery and characterization of O-phosphorylation, this review also briefly outlines the current knowledge about N-phosphorylation depicting its ubiquitous relevance. Further, common pitfalls in sample preparation, LC-MS analysis, and subsequent data analysis are discussed as well as issues regarding quality and comparability of studies on protein phosphorylation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/elps.201200710
  • Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation
    Subramanian, H. and Zahedi, R.P. and Sickmann, A. and Walter, U. and Gambaryan, S.
    Journal of Thrombosis and Haemostasis 11 (2013)
    Background: Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown. Objective: To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation. Methods: The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay. Results: S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation. Conclusion: Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation. © 2013 International Society on Thrombosis and Haemostasis.
    view abstract10.1111/jth.12271
  • Response: platelet transcriptome and proteome--relation rather than correlation.
    Geiger, J. and Burkhart, J.M. and Gambaryan, S. and Walter, U. and Sickmann, A. and Zahedi, R.P.
    Blood 121 (2013)
    view abstract10.1182/blood-2013-04-493403
  • The bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles
    Matkovic, T. and Siebert, M. and Knoche, E. and Depner, H. and Mertel, S. and Owald, D. and Schmidt, M. and Thomas, U. and Sickmann, A. and Kamin, D. and Hell, S.W. and Bürger, J. and Hollmann, C. and Mielke, T. and Wichmann, C. and Sigrist, S.J.
    Journal of Cell Biology 202 (2013)
    Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP. © 2013 Matkovic et al.
    view abstract10.1083/jcb.201301072
  • A complex standard for protein identification, designed by evolution
    Vaudel, M. and Burkhart, J.M. and Breiter, D. and Zahedi, R.P. and Sickmann, A. and Martens, L.
    Journal of Proteome Research 11 (2012)
    Shotgun proteomic investigations rely on the algorithmic assignment of mass spectra to peptides. The quality of these matches is therefore a cornerstone in the analysis and has been the subject of numerous recent developments. In order to establish the benefits of novel algorithms, they are applied to reference samples of known content. However, these were recently shown to be either too simple to resemble typical real-life samples or as leading to results of lower accuracy as the method itself. Here, we describe how to use the proteome of Pyrococcus furiosus, a hyperthermophile, as a standard to evaluate proteomics identification workflows. Indeed, we prove that the Pyrococcus furiosus proteome provides a valid method for detecting random hits, comparable to the decoy databases currently in popular use, but we also prove that the Pyrococcus furiosus proteome goes squarely beyond the decoy approach by also providing many hundreds of highly reliable true positive hits. Searching the Pyrococcus furiosus proteome can thus be used as a unique test that provides the ability to reliably detect both false positives as well as proteome-scale true positives, allowing the rigorous testing of identification algorithms at the peptide and protein level. © 2012 American Chemical Society.
    view abstract10.1021/pr300055q
  • CLP36 is a negative regulator of glycoprotein VI signaling in platelets
    Gupta, S. and Braun, A. and Morowski, M. and Premsler, T. and Bender, M. and Nagy, Z. and Sickmann, A. and Hermanns, H.M. and Bösl, M. and Nieswandt, B.
    Circulation Research 111 (2012)
    RATIONALE: At sites of vascular injury, exposed subendothelial collagens not only trigger sudden platelet adhesion and aggregation, thereby initiating normal hemostasis, but also can lead to acute ischemic diseases, such as myocardial infarction or stroke. The glycoprotein (GP) VI/Fc receptor γ-chain complex is a central regulator of these processes because it mediates platelet activation on collagens through a series of tyrosine phosphorylation events downstream of the Fc receptor γ-chain-associated immunoreceptor tyrosine-based activation motif. GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. OBJECTIVE: We studied the role of PDZ and LIM domain family member CLP36 in platelet physiology in vitro and in vivo. METHODS AND RESULTS: We report that CLP36 acts as a major inhibitor of GPVI immunoreceptor tyrosine-based activation motif signaling in platelets. Platelets from mice either expressing a low amount of a truncated form of CLP36 lacking the LIM domain (Clp36) or lacking the whole protein (Clp36) displayed profound hyperactivation in response to GPVI agonists, whereas other signaling pathways were unaffected. This was associated with hyperphosphorylation of signaling proteins and enhanced Ca mobilization, granule secretion, and integrin activation downstream of GPVI. The lack of functional CLP36 translated into accelerated thrombus formation and enhanced procoagulant activity, assembling a prothrombotic phenotype in vivo. CONCLUSIONS: These data reveal an inhibitory function of CLP36 in GPVI immunoreceptor tyrosine-based activation motif signaling and establish it as a key regulator of arterial thrombosis. © 2012 American Heart Association, Inc.
    view abstract10.1161/CIRCRESAHA.112.264754
  • Current methods for global proteome identification
    Vaudel, M. and Sickmann, A. and Martens, L.
    Expert Review of Proteomics 9 (2012)
    In a time frame of a few decades, protein identification went from laborious single protein identification to automated identification of entire proteomes. This shift was enabled by the emergence of peptide-centric, gel-free analyses, in particular the so-called shotgun approaches, which not only rely on extensive experiments, but also on cutting-edge data processing methods. The present review therefore provides an overview of a shotgun proteomics identification workflow, listing the state-of-the-art methods involved and software that implement these. The authors focus on freely available tools where possible. Finally, data analysis in the context of emerging across-omics studies will also be discussed briefly, where proteomics goes beyond merely delivering a list of protein accession numbers. © 2012 Expert Reviews Ltd.
    view abstract10.1586/epr.12.51
  • DipA, a pore-forming protein in the outer membrane of lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates
    Thein, M. and Bonde, M. and Bunikis, I. and Denker, K. and Sickmann, A. and Bergström, S. and Benz, R.
    PLoS ONE 7 (2012)
    Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species. © 2012 Thein et al.
    view abstract10.1371/journal.pone.0036523
  • E. coli LoiP (YggG), a metalloprotease hydrolyzing Phe-Phe bonds
    Lütticke, C. and Hauske, P. and Lewandrowski, U. and Sickmann, A. and Kaiser, M. and Ehrmann, M.
    Molecular BioSystems 8 (2012)
    YggG is a conserved lipoprotein localized to the outer membrane of Gram negative bacteria. Even though the expressed open reading frame has been identified previously, the Escherichia coli protein remained uncharacterized. We report that YggG of E. coli is a metalloprotease that cleaves its targets preferentially between Phe-Phe residues. Since the yggG promoter is upregulated when bacteria are subjected to media of low osmolarity, YggG was named LoiP (low osmolarity induced protease). LoiP has an intramolecular disulfide (S-S) bond that is formed even in the absence of the periplasmic oxido-reductase DsbA and proper membrane localization of LoiP can depend on another putative metalloprotease, YfgC. © 2012 The Royal Society of Chemistry.
    view abstract10.1039/c2mb05506f
  • Integral quantification accuracy estimation for reporter ion-based quantitative proteomics (iQuARI)
    Vaudel, M. and Burkhart, J.M. and Radau, S. and Zahedi, R.P. and Martens, L. and Sickmann, A.
    Journal of Proteome Research 11 (2012)
    With the increasing popularity of comparative studies of complex proteomes, reporter ion-based quantification methods such as iTRAQ and TMT have become commonplace in biological studies. Their appeal derives from simple multiplexing and quantification of several samples at reasonable cost. This advantage yet comes with a known shortcoming: precursors of different species can interfere, thus reducing the quantification accuracy. Recently, two methods were brought to the community alleviating the amount of interference via novel experimental design. Before considering setting up a new workflow, tuning the system, optimizing identification and quantification rates, etc. one legitimately asks: is it really worth the effort, time and money? The question is actually not easy to answer since the interference is heavily sample and system dependent. Moreover, there was to date no method allowing the inline estimation of error rates for reporter quantification. We therefore introduce a method called iQuARI to compute false discovery rates for reporter ion based quantification experiments as easily as Target/Decoy FDR for identification. With it, the scientist can accurately estimate the amount of interference in his sample on his system and eventually consider removing shadows subsequently, a task for which reporter ion quantification might not be the solution of choice. © 2012 American Chemical Society.
    view abstract10.1021/pr300247u
  • Intermembrane space proteome of yeast mitochondria
    Vögtle, F.-N. and Burkhart, J.M. and Rao, S. and Gerbeth, C. and Hinrichs, J. and Martinou, J.-C. and Chacinska, A. and Sickmann, A. and Zahedi, R.P. and Meisinger, C.
    Molecular and Cellular Proteomics 11 (2012)
    The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
    view abstract10.1074/mcp.M112.021105
  • ITRAQ analysis of a cell culture model for malignant transformation, including comparison with 2D-PAGE and SILAC
    Pütz, S.M. and Boehm, A.M. and Stiewe, T. and Sickmann, A.
    Journal of Proteome Research 11 (2012)
    To study human cancer development, cell culture models for malignant transformation can be used. In 1999 Hahn and Coworkers introduced such a model system and established herewith a basis for research on human tumorigenesis. Primary human fibroblasts are sequentially transduced with defined genetic elements (hTERT, SV40 ER, and H-Ras V12), resulting in four defined cell lines, whereby the last has a fully transformed phenotype. In order to get a deeper insight into the molecular biology of human tumorigenesis, we compared the proteomes of these four cell lines following a multimethod concept. At the beginning we assumed SILAC and sample fractionation with COFRADIC is the method of choice to analyze the cell culture model for malignant transformation. Here, the compared samples are combined before sample preparation, thus avoiding differences in sample preparation, and using COFRADIC notably reduces sample complexity. Because 2D-PAGE is a standard method for the separation and visualization of closely related proteomes, we decided to analyze and compare the proteomes of these four cell lines in a first approach by differential 2D-PAGE. Surprisingly, we discovered much more unique results with iTRAQ and sample fractionation with SCX than with the combination of 2D-PAGE and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies not only in number of yielded results but also in analysis time. Here, we present the iTRAQ quantification results and compare them with the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the protein level at each transition. Thereby, SV40 has the strongest impact on the proteome. In detail we identified 201 regulated proteins. Beside others, these proteins are involved in cytoskeleton, RNA processing, and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins. For example, MCM proteins are up-regulated and collagens are down-regulated due to SV40 ER expression. Furthermore we made the observation that proteins containing the same domain have analogous regulation profiles during malignant transformation. For instance, several proteins containing a CH or LIM domain are down-regulated. Moreover, by this study and the defined cell culture model, changes could be clearly matched to specific steps during tumorigenesis. © 2012 American Chemical Society.
    view abstract10.1021/pr200881c
  • ITRAQ data interpretation
    Vaudel, M. and Burkhart, J.M. and Zahedi, R.P. and Martens, L. and Sickmann, A.
    Methods in Molecular Biology 893 (2012)
    Quantitative proteomic analysis can help elucidating unexplored biological questions; it, however, relies on highly reproducible experiments and reliable data processing. Among the existing strategies, iTRAQ is known as an easy to use method allowing relative comparison of up to eight multiplexed samples. Once the data is acquired it is important that the final protein quantification reflects the actual amounts in the samples. Data interpretation must thus be achieved with a constant focus on quality. Here, we describe a workflow for processing iTRAQ data in user-friendly environments with emphasis on quality control. © 2012 Springer Science+Business Media, LLC.
    view abstract10.1007/978-1-61779-885-6_30
  • Metabolic enzyme diversity in different human thyroid cell lines and their sensitivity to gravitational forces
    Pietsch, J. and Sickmann, A. and Weber, G. and Bauer, J. and Egli, M. and Wildgruber, R. and Infanger, M. and Grimm, D.
    Proteomics 12 (2012)
    Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201200070
  • N-glycoproteomics: Mass spectrometry-based glycosylation site annotation
    Pasing, Y. and Sickmann, A. and Lewandrowski, U.
    Biological Chemistry 393 (2012)
    Glycosylations are ubiquitous and, in many cases, essential protein modifications. Yet comprehensive and detailed analysis of glycosylations on a proteome-wide scale is a daunting and still unsolved challenge. However, a common workflow has emerged over the last decade for large-scale N-glycosylation site annotation by application of proteomic methodology. Thereby, the qualitative and quantitative assessment of hundreds or thousands of modification sites is enabled. This review presents a short overview about common enrichment techniques and glycosylation site detection for N-glycopeptides, including benefits and challenges of analysis. © 2012 by Walter de Gruyter · Berlin · Boston.
    view abstract10.1515/hsz-2011-0245
  • Proteomic and metabolomic analyses of mitochondrial complex I-deficient mouse model generated by spontaneous B2 short interspersed nuclear element (SINE) insertion into NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) gene
    Leong, D.W. and Komen, J.C. and Hewitt, C.A. and Arnaud, E. and McKenzie, M. and Phipson, B. and Bahlo, M. and Laskowski, A. and Kinkel, S.A. and Davey, G.M. and Heath, W.R. and Voss, A.K. and Zahedi, R.P. and Pitt, J.J. and Chrast, R. and Sickmann, A. and Ryan, M.T. and Smyth, G.K. and Thorburn, D.R. and Scott, H.S.
    Journal of Biological Chemistry 287 (2012)
    Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4fky, the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4fky/fkymice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4fky/fky mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "Nassembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/ NAD+ ratio that inhibits mitochondrial fatty acid β-oxidation. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
    view abstract10.1074/jbc.M111.327601
  • Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells
    Sanges, C. and Scheuermann, C. and Zahedi, R.P. and Sickmann, A. and Lamberti, A. and Migliaccio, N. and Baljuls, A. and Marra, M. and Zappavigna, S. and Rapp, U. and Abbruzzese, A. and Caraglia, M. and Arcari, P.
    Cell Death and Disease 3 (2012)
    We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B- and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes. © 2012 Macmillan Publishers Limited All rights reserved.
    view abstract10.1038/cddis.2012.16
  • Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells (vol 3, e276, 2012)
    Sanges, C. and Scheuermann, C. and Zahedi, R. P. and Sickmann, A. and Lamberti, A. and Migliaccio, N. and Baljuls, A. and Marra, M. and Zappavigna, S. and Reinders, J. and Rapp, U. and Abbruzzese, A. and Caraglia, M. and Arcari, P.
    Cell Death & Disease 3 (2012)
    view abstract10.1038/cddis.2012.67
  • Robust workflow for iTRAQ-based peptide and protein quantification
    Beck, F. and Burkhart, J.M. and Geiger, J. and Zahedi, R.P. and Sickmann, A.
    Methods in Molecular Biology 893 (2012)
    Quantitative proteomics has become a routinely used technique to globally compare protein content and expression profiles of biological samples, for instance after differential stimulation. In this context, chemical stable isotope-based labeling techniques, such as ICAT and iTRAQ, have been successfully applied in a large variety of studies. Since iTRAQ labels are isobaric, quantitation is conducted on the MS/MS level. Consequently, up to eight samples can be multiplexed and quantified in a single experiment without increasing sample complexity. Here, we present a robust workflow to conduct iTRAQ quantification of biological samples such as human platelets, which comprises (a) an adequate sample preparation procedure, (b) an optimized tryptic digestion protocol, (c) SPE desalting and subsequent peptide labeling using a 4-plex iTRAQ labeling kit, and (d) fractionation of the obtained peptide mixture by strong cation exchange chromatography. © 2012 Springer Science+Business Media, LLC.
    view abstract10.1007/978-1-61779-885-6_8
  • STEAP1 is associated with the invasive and oxidative stress phenotype of ewing tumors
    Grunewald, T.G.P. and Diebold, I. and Esposito, I. and Plehm, S. and Hauer, K. and Thiel, U. and Da Silva-Buttkus, P. and Neff, F. and Unland, R. and Müller-Tidow, C. and Zobywalski, C. and Lohrig, K. and Lewandrowski, U. and Sickmann, A. and Da Costa, O.P. and Görlach, A. and Cossarizza, A. and Butt, E. and Richter, G.H.S. and Burdach, S.
    Molecular Cancer Research 10 (2012)
    Ewing tumors comprise the second most common type of bone-associated cancer in children and are characterized by oncogenic EWS/FLI1 fusion proteins and early metastasis. Compelling evidence suggests that elevated levels of intracellular oxidative stress contribute to enhanced aggressiveness of numerous cancers, possibly including Ewing tumors. Using comprehensive microarray analyses and RNA interference, we identified the six-transmembrane epithelial antigen of the prostate 1 (STEAP1) - a membrane-bound mesenchymal stem cell marker of unknown function - as a highly expressed protein in Ewing tumors compared with benign tissues and show its regulation by EWS/FLI1. In addition, we show that STEAP1 knockdown reduces Ewing tumor proliferation, anchorage-independent colony formation as well as invasion in vitro and decreases growth and metastasis of Ewing tumor xenografts in vivo. Moreover, transcriptome and proteome analyses as well as functional studies revealed that STEAP1 expression correlates with oxidative stress responses and elevated levels of reactive oxygen species that in turn are able to regulate redox-sensitive and proinvasive genes. In synopsis, our data suggest that STEAP1 is associated with the invasive behavior and oxidative stress phenotype of Ewing tumors and point to a hitherto unanticipated oncogenic function of STEAP1. ©2011 AACR.
    view abstract10.1158/1541-7786.MCR-11-0524
  • Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics
    Burkhart, J.M. and Schumbrutzki, C. and Wortelkamp, S. and Sickmann, A. and Zahedi, R.P.
    Journal of Proteomics 75 (2012)
    Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~. 14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies. © 2011 Elsevier B.V.
    view abstract10.1016/j.jprot.2011.11.016
  • The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways
    Burkhart, J.M. and Vaudel, M. and Gambaryan, S. and Radau, S. and Walter, U. and Martens, L. and Geiger, J. and Sickmann, A. and Zahedi, R.P.
    Blood 120 (2012)
    Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for ∼ 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible. © 2012 by The American Society of Hematology.
    view abstract10.1182/blood-2012-04-416594
  • Well-defined amphiphilic poly(2-oxazoline) ABA-triblock copolymers and their aggregation behavior in aqueous solution
    Krumm, C. and Fik, C.P. and Meuris, M. and Dropalla, G.J. and Geltenpoth, H. and Sickmann, A. and Tiller, J.C.
    Macromolecular Rapid Communications 33 (2012)
    Self-organization of block copolymers in solution is a way to obtain advanced functional superstructures. The synthesis of well-defined polymethyloxazoline-block-polyphenyloxazoline-block-polymethyloxazoline triblock copolymers is described and proven by 1H NMR spectroscopy, SEC, and ESI-MS. The surprisingly water- soluble block copolymers do self-organize in aqueous solutions uniquely forming three coexisting well-defined structures: unimolecular micelles, micellar aggregates, and very form-stable polymersomes. This is the first example of a polymersome forming ABA-triblock copolymer with a glassy middle block. The spherical vesicles are analysed by scanning electron microscopy and transmission electron microscopy. It could be shown that these vesicles are indeed hollow spheres. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/marc.201200192
  • A proteomic approach to analysing spheroid formation of two human thyroid cell lines cultured on a random positioning machine
    Pietsch, J. and Sickmann, A. and Weber, G. and Bauer, J. and Egli, M. and Wildgruber, R. and Infanger, M. and Grimm, D.
    Proteomics 11 (2011)
    The human cell lines FTC-133 and CGTH W-1, both derived from patients with thyroid cancer, assemble to form different types of spheroids when cultured on a random positioning machine. In order to obtain a possible explanation for their distinguishable aggregation behaviour under equal culturing conditions, we evaluated a proteomic analysis emphasising cytoskeletal and membrane-associated proteins. For this analysis, we treated the cells by ultrasound, which freed up some of the proteins into the supernatant but left some attached to the cell fragments. Both types of proteins were further separated by free-flow IEF and SDS gel electrophoresis until their identity was determined by MS. The MS data revealed differences between the two cell lines with regard to various structural proteins such as vimentin, tubulins and actin. Interestingly, integrin α-5 chains, myosin-10 and filamin B were only found in FTC-133 cells, while collagen was only detected in CGTH W-1 cells. These analyses suggest that FTC-133 cells express surface proteins that bind fibronectin, strengthening the three-dimensional cell cohesion. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000817
  • Catch me if you can: Mass spectrometry-based phosphoproteomics and quantification strategies
    Eyrich, B. and Sickmann, A. and Zahedi, R.P.
    Proteomics 11 (2011)
    Phosphorylation of proteins is one of the most prominent PTMs and for instance a key regulator of signal transduction. In order to improve our understanding of cellular phosphorylation events, considerable effort has been devoted to improving the analysis of phosphorylation by MS-based proteomics. Different enrichment strategies for phosphorylated peptides/proteins, such as immunoaffinity chromatography (IMAC) or titanium dioxide, have been established and constantly optimized for subsequent MS analysis. Concurrently, specific MS techniques were developed for more confident identification and phosphorylation site localization. In addition, more attention is paid to the LC-MS instrumentation to avoid premature loss of phosphorylated peptides within the analytical system. Despite major advances in all of these fields, the analysis of phosphopeptides still remains far from being routine in proteomics. However, to reveal cellular regulation by phosphorylation events, not only qualitative information about the phosphorylation status of proteins but also, in particular, quantitative information about distinct changes in phosphorylation patterns upon specific stimulation is mandatory. Thus, yielded insights are of outstanding importance for the emerging field of systems biology. In this review, we will give an insight into the historical development of phosphoproteome analysis and discuss its recent progress particularly regarding phosphopeptide quantification and assessment of phosphorylation stoichiometry. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000489
  • Compomics-utilities: An open-source Java library for computational proteomics
    Barsnes, H. and Vaudel, M. and Colaert, N. and Helsens, K. and Sickmann, A. and Berven, F.S. and Martens, L.
    BMC Bioinformatics 12 (2011)
    Background: The growing interest in the field of proteomics has increased the demand for software tools and applications that process and analyze the resulting data. And even though the purpose of these tools can vary significantly, they usually share a basic set of features, including the handling of protein and peptide sequences, the visualization of (and interaction with) spectra and chromatograms, and the parsing of results from various proteomics search engines. Developers typically spend considerable time and effort implementing these support structures, which detracts from working on the novel aspects of their tool.Results: In order to simplify the development of proteomics tools, we have implemented an open-source support library for computational proteomics, called compomics-utilities. The library contains a broad set of features required for reading, parsing, and analyzing proteomics data. compomics-utilities is already used by a long list of existing software, ensuring library stability and continued support and development.Conclusions: As a user-friendly, well-documented and open-source library, compomics-utilities greatly simplifies the implementation of the basic features needed in most proteomics tools. Implemented in 100% Java, compomics-utilities is fully portable across platforms and architectures. Our library thus allows the developers to focus on the novel aspects of their tools, rather than on the basic functions, which can contribute substantially to faster development, and better tools for proteomics. © 2011 Barsnes et al; licensee BioMed Central Ltd.
    view abstract10.1186/1471-2105-12-70
  • Composition and topology of the endoplasmic reticulum-mitochondria encounter structure
    Stroud, D.A. and Oeljeklaus, S. and Wiese, S. and Bohnert, M. and Lewandrowski, U. and Sickmann, A. and Guiard, B. and Van Der Laan, M. and Warscheid, B. and Wiedemann, N.
    Journal of Molecular Biology 413 (2011)
    Eukaryotic cells contain multiple organelles, which are functionally and structurally interconnected. The endoplasmic reticulum-mitochondria encounter structure (ERMES) forms a junction between mitochondria and the endoplasmic reticulum (ER). Four ERMES proteins are known in yeast, the ER-anchored protein Mmm1 and three mitochondria-associated proteins, Mdm10, Mdm12 and Mdm34, with functions related to mitochondrial morphology and protein biogenesis. We mapped the glycosylation sites of ERMES and demonstrate that three asparagine residues in the N terminal domain of Mmm1 are glycosylated. While the glycosylation is dispensable, the cytosolic C terminal domain of Mmm1 that connects to the Mdm proteins is required for Mmm1 function. To analyze the composition of ERMES, we determined the subunits by quantitative mass spectrometry. We identified the calcium-binding GTPase Gem1 as a new ERMES subunit, revealing that ERMES is composed of five genuine subunits. Taken together, ERMES represents a platform that integrates components with functions in formation of ER-mitochondria junctions, maintenance of mitochondrial morphology, protein biogenesis and calcium binding. © 2011 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.jmb.2011.09.012
  • Dual function of Sdh3 in the respiratory Chain and TIM22 protein translocase of the mitochondrial inner membrane
    Gebert, N. and Gebert, M. and Oeljeklaus, S. and von der Malsburg, K. and Stroud, D.A. and Kulawiak, B. and Wirth, C. and Zahedi, R.P. and Dolezal, P. and Wiese, S. and Simon, O. and Schulze-Specking, A. and Truscott, K.N. and Sickmann, A. and Rehling, P. and Guiard, B. and Hunte, C. and Warscheid, B. and van der Laan, M. and Pfanner, N. and Wiedemann, N.
    Molecular Cell 44 (2011)
    The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively. © 2011 Elsevier Inc.
    view abstract10.1016/j.molcel.2011.09.025
  • Identification of Eps15 as antigen recognized by the monoclonal antibodies aa2 and ab52 of the wuerzburg hybridoma library against Drosophila brain
    Halder, P. and Chen, Y.-c. and Brauckhoff, J. and Hofbauer, A. and Dabauvalle, M.-C. and Lewandrowski, U. and Winkler, C. and Sickmann, A. and Buchner, E.
    PLoS ONE 6 (2011)
    The Wuerzburg Hybridoma Library against the Drosophilabrain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies. © 2011 Halder et al.
    view abstract10.1371/journal.pone.0029352
  • iTRAQ protein quantification: A quality-controlled workflow
    Burkhart, J.M. and Vaudel, M. and Zahedi, R.P. and Martens, L. and Sickmann, A.
    Proteomics 11 (2011)
    Reporter ion-based methods are among the major techniques to quantify peptides and proteins. Two main labels, tandem mass tag (TMT) and iTRAQ, are widely used by the proteomics community. They are, however, often applied as out-of-the-box methods, without thorough quality control. Thus, due to undiscovered limitations of the technique, irrelevant results might be trusted. To address this issue, we here propose a step-by-step quality control of the iTRAQ workflow. From sample preparation to final ratio calculation we provide metrics and techniques assessing the actual effectiveness of iTRAQ quantification as well as a novel method for more reliable protein ratio estimation. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000711
  • Mass-Spectrometry-Based Proteomics: General Overview and Posttranslational Modification Analysis in the Context of Platelet Research
    Maria Burkhart, J. and Sickmann, A.
    Platelet Proteomics: Principles, Analysis, and Applications (2011)
    In recent years, mass spectrometry-based proteomics has become an indispensable tool allowing the analysis of protein patterns and sensitive changes of post-translational modifications (PTMs). Mass spectrometry has been increasingly applied for platelet research, thereby enabling the analysis of functionally relevant processes on a molecular level, especially with regard to PTM-regulated activation/inhibition. In this chapter a general overview including the analysis of PTM is presented and respective platelet studies are discussed. © 2011 John Wiley & Sons, Inc.
    view abstract10.1002/9780470940297.ch2
  • Peptide identification quality control
    Vaudel, M. and Burkhart, J.M. and Sickmann, A. and Martens, L. and Zahedi, R.P.
    Proteomics 11 (2011)
    Identification of large proteomics data sets is routinely performed using sophisticated software tools called search engines. Yet despite the importance of the identification process, its configuration and execution is often performed according to established lab habits, and is mostly unsupervised by detailed quality control. In order to establish easily obtainable quality control criteria that can be broadly applied to the identification process, we here introduce several simple quality control methods. An unbiased quality control of identification parameters will be conducted using target/decoy searches providing significant improvement over identification standards. MASCOT identifications were for instance increased by 13% at a constant level of confidence. The target/decoy approach can however not be universally applied. We therefore also quality control the application of this strategy itself, providing useful and intuitive metrics for evaluating the precision and robustness of the obtained false discovery rate. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000704
  • Phosphoproteome analysis of the platelet plasma membrane
    Premsler, T. and Lewandrowski, U. and Sickmann, A. and Zahedi, R.P.
    Methods in Molecular Biology 728 (2011)
    Blood platelets are key players standing at the crossroads between physiologically occurring hemostasis and pathologic thrombus formation. As these cellular particles lack a nucleus, intra- and intercellular processes involved in platelet activity and function are almost exclusively regulated on the protein level. In particular, posttranslational protein modification by phosphorylation, which allows for a quick and highly dynamic transduction of cellular signals, is discussed in this context. In addition, since platelet activation and aggregation usually require surface contact with the surrounding tissue, special interest focuses on this contacting region, and hence on the subproteome of the platelet plasma membrane. In this chapter, we present a mass spectrometry-driven approach capable of dealing with the task of platelet plasma membrane proteomics and phosphoproteomics. The outlined protocols include strategies for the isolation and purification of plasma membrane proteins by aqueous two-phase partitioning and subsequent enrichment of phosphopeptides via titanium dioxide chromatography. © 2011 Springer Science+Business Media, LLC.
    view abstract10.1007/978-1-61779-068-3_19
  • Quality control of nano-LC-MS systems using stable isotope-coded peptides
    Burkhart, J.M. and Premsler, T. and Sickmann, A.
    Proteomics 11 (2011)
    In analytical sciences, there is a general need for quality control to assess whether a product or a process meets defined requirements. Especially in proteomics, which implies analysis of ten thousands of analytes within a complex mixture, quality control to validate LC-MS performance and method setup is inevitable to achieve day-to-day-, inter-system-, as well as inter-user reproducibility. Thus, results deriving from LC-MS analyses can be benchmarked and the need for system maintenance can be revealed. In particular with the advent of label-free quantification of peptides and proteins, which above all depends on highly stable and reproducible LC separations, HPLC performance has to be appropriately monitored throughout the entire analytical procedure to assure quality and validity of the obtained data. Oftentimes, proteolytic digests of standard proteins are used in this context; however, this approach implies some limitations, such as inadequate batch-to-batch reproducibility, limited (if any) dynamic range and compositional inflexibility. Here, we present an alternative strategy of nano-LC-MS/MS quality control based on a mixture of synthetic peptides covering the entire LC-gradient as well as a dynamic range of more than two orders of magnitude. Thus, (i) reproducibility of LC separation, (ii) MS performance (including limit of detection, identification and quantification), as well as (iii) overall nano-LC-MS system performance and reproducibility can be routinely monitored even in highly complex samples. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000604
  • Regulation of mitochondrial protein import by cytosolic kinases
    Schmidt, O. and Harbauer, A.B. and Rao, S. and Eyrich, B. and Zahedi, R.P. and Stojanovski, D. and Schönfisch, B. and Guiard, B. and Sickmann, A. and Pfanner, N. and Meisinger, C.
    Cell 144 (2011)
    Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases - in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria. © 2011 Elsevier Inc.
    view abstract10.1016/j.cell.2010.12.015
  • RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity
    Guderian, G. and Peter, C. and Wiesner, J. and Sickmann, A. and Schulze-Osthoff, K. and Fischer, U. and Grimmler, M.
    Journal of Biological Chemistry 286 (2011)
    Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
    view abstract10.1074/jbc.M110.148486
  • SearchGUI: An open-source graphical user interface for simultaneous OMSSA and X!Tandem searches
    Vaudel, M. and Barsnes, H. and Berven, F.S. and Sickmann, A. and Martens, L.
    Proteomics 11 (2011)
    The identification of proteins by mass spectrometry is a standard technique in the field of proteomics, relying on search engines to perform the identifications of the acquired spectra. Here, we present a user-friendly, lightweight and open-source graphical user interface called SearchGUI, for configuring and running the freely available OMSSA (open mass spectrometry search algorithm) and X!Tandem search engines simultaneously. Freely available under the permissible Apache2 license, SearchGUI is supported on Windows, Linux and OSX. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000595
  • Strong cation exchange chromatography for analysis of sialylated glycopeptides
    Lohrig, K. and Sickmann, A. and Lewandrowski, U.
    Methods in Molecular Biology 753 (2011)
    Glycosylations represent major and essential co- and post-translational modification forms of proteins and facilitate a multitude of functions such as cell-cell interactions as well as protein folding and stability. The analysis of protein glycosylation is still an enormous task due to the vast heterogeneity and multi-tude of different possible carbohydrate structures. The elucidation of glycosylation sites - the attachment points of carbohydrate structures to the polypeptide backbone - is often among the first necessary steps of analysis. Therefore, we here present a simple protocol for charge-based enrichment of sialylated glycopeptides by strong cation exchange chromatography and subsequent analysis of glycosylation sites by mass spectrometry. © Springer Science+Business Media, LLC 2011.
    view abstract10.1007/978-1-61779-148-2_20
  • Subcellular analysis of the platelet proteome
    Premsler, T. and Lewandrowski, U. and Zahedi, R.P. and Sickmann, A.
    Current Proteomics 8 (2011)
    Blood platelets are key players mainly involved in physiological thrombus formation as well as pathological events, such as atherosclerosis, coronary artery disease, or myocardial infarction. Consequently, numerous studies have focused on gaining insight into the platelet proteome, with biomolecular mass spectrometry providing an efficient technology to cope with this task as it nowadays enables both protein identification and differential quantification of cellular material. To assemble a comprehensive proteomic dataset, platelet-centric studies have to be performed not only on the global scale, but also on the subcellular level, which implies subfractionation techniques and targeted approaches prior to detailed proteomic analyses. In this review we present an overview of advances in platelet subcellular proteomics focusing on the platelet membrane proteome and the protein content of both platelet granules and platelet-derived microparticles. Since posttranslational modifications are furthermore known to decisively participate in platelet function e.g. upon activation, analytical methods for the platelet phospho- as well as the glycoproteome will also be discussed. © 2011 Bentham Science Publishers.
    view abstract10.2174/157016411797247440
  • The good, the bad, the ugly: Validating the mass spectrometric analysis of modified peptides
    Beck, F. and Lewandrowski, U. and Wiltfang, M. and Feldmann, I. and Geiger, J. and Sickmann, A. and Zahedi, R.P.
    Proteomics 11 (2011)
    Mass spectrometric characterization of protein modifications is usually based on single peptides. With the advent of large-scale PTM-focussed MS studies, vast amounts of data are generated continuously, providing biologists extremely valuable and virtually never-ending sources for targeted functional research. However, even more than for proteomics in general, appropriate strategies for quality control of the different steps of the analytical strategy are imperative to prevent functional researchers from doing Sisyphos work on false-positive and unconfident PTM assignments. Here, we describe strategies to address the important issue of quality control for PTM analysis on various levels of the analytical pipeline: sample preparation/processing, analysis/identification and finally data interpretation, for qualitative as well as quantitative studies. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000562
  • Thermo-msf-parser: An open source Java library to parse and visualize Thermo Proteome Discoverer msf files
    Colaert, N. and Barsnes, H. and Vaudel, M. and Helsens, K. and Timmerman, E. and Sickmann, A. and Gevaert, K. and Martens, L.
    Journal of Proteome Research 10 (2011)
    The Thermo Proteome Discoverer program integrates both peptide identification and quantification into a single workflow for peptide-centric proteomics. Furthermore, its close integration with Thermo mass spectrometers has made it increasingly popular in the field. Here, we present a Java library to parse the msf files that constitute the output of Proteome Discoverer. The parser is also implemented as a graphical user interface allowing convenient access to the information found in the msf files, and in Rover, a program to analyze and validate quantitative proteomics information. All code, binaries, and documentation is freely available at http://thermo-msf-parser.googlecode. com. © 2011 American Chemical Society.
    view abstract10.1021/pr2005154
  • A stimulatory role for the La-related protein 4B in translation
    Schäffler, K. and Schulz, K. and Hirmer, A. and Wiesner, J. and Grimm, M. and Sickmann, A. and Fischer, U.
    RNA 16 (2010)
    La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3′ end with initiating ribosomes. Copyright © 2010 RNA Society.
    view abstract10.1261/rna.2146910
  • Application of free-flow IEF to identify protein candidates changing under microgravity conditions
    Pietsch, J. and Kussian, R. and Sickmann, A. and Bauer, J. and Weber, G. and Nissum, M. and Westphal, K. and Egli, M. and Grosse, J. and Schönberger, J. and Wildgruber, R. and Infanger, M. and Grimm, D.
    Proteomics 10 (2010)
    Using antibody-related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s-μg). In this study, we performed proteome analysis in order to identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-μg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free-flow IEF. The fractions obtained were further separated by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free-flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s-μg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/pmic.200900226
  • Characterization of a novel interaction between vasodilator-stimulated phosphoprotein and abelson interactor 1 in human platelets: A concerted computational and experimental approach
    Dittrich, M. and Strassberger, V. and Fackler, M. and Tas, P. and Lewandrowski, U. and Sickmann, A. and Walter, U. and Dandekar, T. and Birschmann, I.
    Arteriosclerosis, Thrombosis, and Vascular Biology 30 (2010)
    Objective: The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. Methods and results: Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum., we performed a motif search of all sequences and identified potential target sites of class I EVH1 domains in human platelet proteins. Performing affinity purification with VASP-EVH1 domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 domain-specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. Conclusion: The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, as they offer the potential to analyze signal transduction beyond the scope of established pathways. © 2010 American Heart Association, Inc.
    view abstract10.1161/ATVBAHA.109.200683
  • Comparative proteomic analysis of biofilm and planktonic cells of Neisseria meningitidis
    Van Alen, T. and Claus, H. and Zahedi, R.P. and Groh, J. and Blazyca, H. and Lappann, M. and Sickmann, A. and Vogel, U.
    Proteomics 10 (2010)
    Neisseria meningitidis is a commensal of the human nasopharynx occasionally causing invasive disease. In vitro biofilms have been employed to model meningococcal carriage. A proteomic analysis of meningococcal biofilms was conducted and metabolic changes related to oxygen and nutrient limitation and upregulation of proteins involved in ROS defense were observed. The upregulated MntC which protects against ROS was shown to be required for meningococcal biofilm formation, but not for planktonic growth. ROS-induced proteomic changes might train the biofilm to cope with immune effectors. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/pmic.201000267
  • Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern
    Schröter, J. and Zahedi, R.P. and Hartmann, M. and Gaßner, B. and Gazinski, A. and Waschke, J. and Sickmann, A. and Kuhn, M.
    FEBS Journal 277 (2010)
    Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3′,5′-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications. © 2010 FEBS.
    view abstract10.1111/j.1742-4658.2010.07658.x
  • Identification of a Protein Kinase C-dependent phosphorylation site involved in sensitization of TRPV4 channel
    Peng, H. and Lewandrowski, U. and Müller, B. and Sickmann, A. and Walz, G. and Wegierski, T.
    Biochemical and Biophysical Research Communications 391 (2010)
    Transient Receptor Potential (TRP) proteins are non-selective cation channels performing diverse cellular functions. TRPV1 and TRPV4, two calcium-permeable channels of the vanilloid subfamily of TRP proteins, are activated by various physical and chemical stimuli, including noxious heat and mechanical stress, respectively. These channels are also required for exaggerated sensation of painful stimuli, condition referred to as hyperalgesia, which is frequently associated with inflammation. Phosphorylation of TRPV1, involving Protein Kinase C (PKC) and Protein Kinase A (PKA), appears to be the predominant mechanism for channel sensitization and development of heat hyperalgesia. PKC and PKA pathways have also been implicated in the sensitization of TRPV4, but the respective phosphorylation sites remain unknown. Using mass spectrometry, we report now that TRPV4 is phosphorylated on serine 824 by the PKC-activating phorbol 12-myristate 13-acetate. This phosphorylation is prevented by a PKC inhibitor, confirming the involvement of PKC. Ser824, located in the carboxy-terminal cytosolic tail of TRPV4, is also phosphorylated after activation of the PKA pathway by forskolin, albeit less potently. Substitution of Ser824 with aspartic acid, mimicking phosphorylation at this site, increased TRPV4-mediated calcium influx in resting and in stimulated cells, underlining the importance of this residue in TRPV4 regulation. Thus PKC, and possibly PKA, phosphorylate TRPV4 at Ser824 leading to the enhancement of TRPV4 channel function. Our findings suggest an important role of this phosphorylation in TRPV4 sensitization and the development of hyperalgesia. © 2009 Elsevier Inc. All rights reserved.
    view abstract10.1016/j.bbrc.2009.12.140
  • jTraqX: A free, platform independent tool for isobaric tag quantitation at the protein level
    Muth, T. and Keller, D. and Puetz, S.M. and Martens, L. and Sickmann, A. and Boehm, A.M.
    Proteomics 10 (2010)
    Many proteomic studies focus on quantitative aspects, using different stable isotope labeling techniques that require specialized software to analyze the generated data. Here we present jTraqX, an easy-to-use tool for processing and visualizing protein quantification data. jTraqX is platform independent and is compatible with all available 4-plex isobaric tags. jTraqX can be freely downloaded at © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/pmic.200900374
  • Malignant transformation in a defined genetic background: Proteome changes displayed by 2D-PAGE
    Pütz, S.M. and Vogiatzi, F. and Stiewe, T. and Sickmann, A.
    Molecular Cancer 9 (2010)
    Background: Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).Results: The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot.Conclusion: These findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis. © 2010 Pütz et al; licensee BioMed Central Ltd.
    view abstract10.1186/1476-4598-9-254
  • ms-lims, a simple yet powerful open source laboratory information management system for MS-driven proteomics
    Helsens, K. and Colaert, N. and Barsnes, H. and Muth, T. and Flikka, K. and Staes, A. and Timmerman, E. and Wortelkamp, S. and Sickmann, A. and Vandekerckhove, J. and Gevaert, K. and Martens, L.
    Proteomics 10 (2010)
    MS-based proteomics produces large amounts of mass spectra that require processing, identification and possibly quantification before interpretation can be undertaken. High-throughput studies require automation of these various steps, and management of the data in association with the results obtained. We here present ms-lims (, a freely available, open-source system based on a central database to automate data management and processing in MS-driven proteomics analyses. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/pmic.200900409
  • Online dual gradient reversed-phase/porous graphitized carbon nanoHPLC for proteomic applications
    Lewandrowski, U. and Sickmann, A.
    Analytical Chemistry 82 (2010)
    The analysis of proteolytic peptide mixtures is among the dominant tasks within proteomic workflows. In order to limit undersampling effects during mass spectrometric detection, online-coupled liquid chromatography is the method of choice, with reversed-phase chromatography being the most important separation mode. Since hydrophilic compounds such as short peptides and some glycosylated species as well as oligosaccharides from glycoproteomic workflows are commonly not accessible by this analytical setup, we hereby present a dual gradient system combining reversed-phase and porous graphitic carbon retention modes within a single nanoHPLC setup. Samples in the low femtomole range are analyzed consecutively first by reversed phase, and nonretained molecules are directly separated by porous graphitic carbon. Both gradient elution systems allow for online coupled mass spectrometric detection and are demonstrated to enable analysis of protein, peptide, and oligosaccharide mixtures within the same setup. Thereby, the accessible range for proteomic and glycoproteomic applications may be extended far beyond the limits of conventional reversed-phase nanoHPLC setups. © 2010 American Chemical Society.
    view abstract10.1021/ac100853w
  • Peptide and protein quantification: A map of the minefield
    Vaudel, M. and Sickmann, A. and Martens, L.
    Proteomics 10 (2010)
    The increasing popularity of gel-free proteomics technologies has created a strong demand for compatible quantitative analysis methods. As a result, a plethora of different techniques has been proposed to perform gel-free quantitative analysis of proteomics samples. Each of these methods comes with certain strengths and shortcomings, and they often are dedicated to a specific purpose. This review will present a brief overview of the main methods, organized by their underlying concepts, and will discuss the issues they raise with a focus on data processing. Finally, we will list the available software that can help with the data processing from quantitative experiments. We hope that this review will thus enable researchers to find the most appropriate method available for their research objectives, and can also serve as a basis for creating a reliable data processing strategy.
    view abstract10.1002/pmic.200900481
  • Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer
    Cadenas, C. and Franckenstein, D. and Schmidt, M. and Gehrmann, M. and Hermes, M. and Geppert, B. and Schormann, W. and Maccoux, L.J. and Schug, M. and Schumann, A. and Wilhelm, C. and Freis, E. and Ickstadt, K. and Rahnenführer, J. and Baumbach, J.I. and Sickmann, A. and Hengstler, J.G.
    Breast Cancer Research 12 (2010)
    Introduction: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets.Methods: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription.Results: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species.Conclusions: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner. © 2010 Cadenas et al.; licensee BioMed Central Ltd.
    view abstract10.1186/bcr2599
  • The Hsp70 chaperone Ssa1 is essential for catabolite induced degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase
    Juretschke, J. and Menssen, R. and Sickmann, A. and Wolf, D.H.
    Biochemical and Biophysical Research Communications 397 (2010)
    Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of gluconeogenesis. In the yeast Saccharomyces cerevisiae, it is only expressed when cells are grown in medium with nonfermentable carbon sources. Addition of glucose to cells leads to inactivation of FBPase and degradation via the ubiquitin-proteasome system. Polyubiquitination of FBPase is carried out by the Gid complex, a multi-subunit ubiquitin ligase. Using tandem affinity purification and subsequent mass spectrometry we identified the Hsp70 chaperone Ssa1 as a novel interaction partner of FBPase. Studies with the temperature-sensitive mutant ssa1-45ts showed that Ssa1 is essential for polyubiquitination of FBPase by the Gid complex. Moreover, we show that degradation of an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is also affected in ssa1-45ts cells demonstrating that Ssa1 plays a general role in elimination of gluconeogenic enzymes. © 2010 Elsevier Inc.
    view abstract10.1016/j.bbrc.2010.05.123
  • XTandem Parser: An open-source library to parse and analyse X!Tandem MS/MS search results
    Muth, T. and Vaudel, M. and Barsnes, H. and Martens, L. and Sickmann, A.
    Proteomics 10 (2010)
    Identification of proteins by MS plays an important role in proteomics. A crucial step concerns the identification of peptides from MS/MS spectra. The X!Tandem Project ( supplies an open-source search engine for this purpose. In this study, we present an open-source Java library called XTandem Parser that parses X!Tandem XML result files into an easily accessible and fully functional object model (http://xtandem-parser.googlecode. com). In addition, a graphical user interface is provided that functions as a usage example and an end-user visualization tool. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA.
    view abstract10.1002/pmic.200900759

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