Research Platelet Membrane Proteomics
Total Page：16
File Type：pdf, Size：1020Kb
From bloodjournal.hematologylibrary.org at GRAND VALLEY STATE UNIV on November 19, 2013. For personal use only. 2009 114: e10-e19 Prepublished online May 12, 2009; doi:10.1182/blood-2009-02-203828 Platelet membrane proteomics: a novel repository for functional research Urs Lewandrowski, Stefanie Wortelkamp, Katharina Lohrig, René P. Zahedi, Dirk A. Wolters, Ulrich Walter and Albert Sickmann Updated information and services can be found at: http://bloodjournal.hematologylibrary.org/content/114/1/e10.full.html Articles on similar topics can be found in the following Blood collections e-Blood (113 articles) Platelets and Thrombopoiesis (407 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From bloodjournal.hematologylibrary.org at GRAND VALLEY STATE UNIV on November 19, 2013. For personal use only. PLATELETS AND THROMBOPOIESIS e-Blood Platelet membrane proteomics: a novel repository for functional research Urs Lewandrowski,1 Stefanie Wortelkamp,1 Katharina Lohrig,2 Rene´P. Zahedi,1 Dirk A. Wolters,2 Ulrich Walter,3 and Albert Sickmann1,4 1Institute for Analytical Sciences (ISAS), Dortmund; 2Department of Analytical Chemistry, Ruhr-University-Bochum, Bochum; 3Institute for Clinical Biochemistry and Pathobiochemistry and Central Laboratory, University Wu¨rzburg, Wu¨rzburg; and 4Medizinisches Proteom-Center (MPC), Ruhr-University-Bochum, Bochum, Germany Being central players in thrombosis and are not accessible to large-scale genomic provides a brief overview of gene ontol- hemostasis, platelets react in manifold screens and thus a comprehensive inven- ogy subcellular and functional classifica- and complex ways to extracellular stimuli. tory of membrane proteins is still miss- tion, as well as interaction network analy- Cell-matrix and cell-cell interactions are ing. For this reason, we here present an sis. In addition, the mass spectrometric mandatory for initial adhesion as well as advanced proteomic setup for the de- data were used to assemble a first tenta- for final development of stable plugs. tailed analysis of enriched platelet plasma tive relative quantification of large-scale Primary interfaces for interactions are membranes and the so far most complete data on the protein level. We therefore plasma membrane proteins, of which collection of platelet membrane proteins. estimate the presented data to be of ma- many have been identified over the past In summary, 1282 proteins were identi- jor interest to the platelet research field decades in individual studies. However, fied, of which more than half are termed and to support rational design of func- due to their enucleate structure, platelets to be of membrane origin. This study tional studies. (Blood. 2009;114:e10-e19) Introduction Platelets are essential mediators of hemostasis and are well known for brane purifications in combination with suitable separation tech- their major role in thrombotic events. In the context of increasing niques,1 which achieved identification of nearly 300 proteins, of numbers of cardiovascular diseases, platelets are of premier interest for which approximately half were of membrane origin. Of these, G6B scientific research. Owing to their unique enucleate ultrastructure (still was later confirmed by Senis et al to be a novel immunoreceptor including organelles such as Golgi, endoplasmic reticulum, or mitochon- tyrosine-based inhibitory motif protein4 as part of an additional dria), common molecular biology–based methods can hardly be applied. study on the platelet membrane proteome. Still, only 46 plasma Their megakaryocyte-derived maturation process renders platelets incon- membrane components could be observed within a total of venient targets for genome-based research approaches. Although mouse 136 membrane proteins in this study, thereby indicating a potential knockout models were generated for a range of functional studies, they gap of knowledge regarding protein membrane constituents. Plasma are limited mostly to known protein components of the platelet system, membrane proteins are among the key targets for platelet drug and estimated to be of functional importance. Moreover, protein synthesis in functional research, since they are the primary interface to extracel- platelets is limited, and a direct correlation between mRNAprofiling and lular stimuli and therefore also for pharmaceutical treatment of protein presence is problematic at best. Based on these limitations, platelet-related disorders. We therefore sought to increase the modern proteome analysis might be a key asset for the analysis of the identification rate of platelet membrane proteins with a major focus platelet proteome and its so far unknown components. The derived on the plasma membrane. knowledge of these newly identified proteins represents a rich source for For plasma membrane enrichment, several methods have been the rational design of functional studies. established in the past, with density gradients being the most Despite their major functional importance a complete inventory common. In addition, lectins have been used to additionally purify of the platelet proteome is far from being accessible. Due to the plasma membrane glycoproteins.1 However, due to our experience development of biomolecular mass spectrometry, a series of with efficient enrichment of rat brain plasma membranes by medium- to large-scale studies was conducted upon the platelet 2-phase aqueous partitioning systems,5,6 we adopted this technique proteome. Early studies aiming for a complete overview of platelet for platelet membranes. Two-phase partitioning is based on sorting proteins most often used 2-dimensional polyacrylamide gel electro- of vesicles upon their physicochemical surface properties (such as phoresis (2D-PAGE) as primary separation technique, frequently in hydrophilicity/hydrophobicity and net surface charge, possibly due conjunction with peptide mass fingerprint identification of proteins. to their phospholipid composition)7 within a defined 2-polymer However, as demonstrated by Moebius et al1 2D-PAGE–based system. A common system is the polyethyleneglycol (PEG)/ studies of complete platelets2,3 were reasonably unsuccessful in dextran system, where plasma membranes show the highest affinity elucidating membrane components compared with targeted mem- for the more hydrophobic upper PEG phase in comparison with Submitted February 4, 2009; accepted April 30, 2009. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, May 12, 2009; DOI 10.1182/blood-2009-02-203828. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. The online version of this article contains a data supplement. © 2009 by The American Society of Hematology e10 BLOOD, 2 JULY 2009 ⅐ VOLUME 114, NUMBER 1 From bloodjournal.hematologylibrary.org at GRAND VALLEY STATE UNIV on November 19, 2013. For personal use only. BLOOD, 2 JULY 2009 ⅐ VOLUME 114, NUMBER 1 PLATELET MEMBRANE PROTEOME e11 other membrane vesicles.8 We already successfully used this 1D-PAGE and digests technique for analysis of plasma membrane N-glycosylation sites Platelet membrane pellets from 2-phase partitioning systems were reconsti- 9,10 on human platelets. In contrast, the current study intends to tuted in 1 ϫ LDS-sample buffer (Invitrogen). After incubation at 75°C for provide a general overview of membrane proteins within these 15 minutes, samples were applied to 4% to 12% Bis-Tris gels using a preparations to allow for a more global assessment of the mem- MOPS buffer system (NuPAGE-Novex; Invitrogen). Protein separation was brane protein composition. followed by colloidal Coomassie staining.15 Subsequently, gel lanes were Here, 3 major strategies were pursued for protein identification: cut in 1-mm slices and bands were treated as described previously.5 After (1) For a global analysis, separation of proteins by 1-dimensional reduction and alkylation, proteins were tryptically digested in-gel and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (1D- peptides were extracted by 0.1% trifluoroacetic acid. SDS-PAGE) followed by nano–liquid chromatography (LC)– tandem mass spectrometry detection of peptides, basically as COFRADIC 6 described previously was conducted. (2) To address issues com- A detailed description of COFRADIC experimental procedures is given in monly encountered with PAGE separation of membrane proteins, the supplemental methods. highly complex peptide mixtures from membrane proteins were separated by strong cation exchange and reversed-phase chromatog- Mass spectrometry raphy within a MudPIT setup (Multidimensional Protein Identifica- tion Technology) prior to mass spectrometric detection.11 (3) To Nano-LC-tandem mass spectrometry of 1D-PAGE and CO- reduce the high complexity of peptide mixtures prior detection, FRADIC fractions. Tryptic peptides