Knowledge of the identity and quantity of expressed proteins of a cell type is a prerequisite for a complete understanding of its molecular functions. Mass-spectrometry-based proteomics has allowed the identification of the entire protein complement of yeast and the close-to-complete set of proteins expressed in mammalian cell lines. Using recent technological advances, we here characterized the proteome of murine platelets, key actors in mediating hemostasis and thrombosis. We accurately measured the absolute protein concentrations of 13 platelet proteins using SILAC-protein epitope signature tags and used them as reference points to estimate the copy numbers of all proteins of the platelet proteome. To distinguish contaminants such as plasma or erythrocyte proteins from true platelet proteins, we monitored protein abundance profiles across multiple purification steps. In total, we absolutely quantified 4,400 platelet proteins, with estimated copy numbers ranging from less than 10 to about a million per cell. Stoichiometries derived from our data correspond well with previous studies. Our study provides a close-to-complete reference map of platelet proteins that will be useful to the community, for instance, for interpreting mouse models of human platelet diseases.
Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.
Mass spectrometry-based proteomics increasingly relies on relative or absolute quantification. In relative quantification, stable isotope based methods often allow mixing at early stages of sample preparation, whereas for absolute quantification this has generally required recombinant expression of full length, labeled protein standards. Here we make use of a very large library of Protein Epitope Signature Tags (PrESTs) that has been developed in the course of the Human Protein Atlas Project. These PrESTs are expressed recombinantly in E. coli and they consist of a short and unique region of the protein of interest as well as purification and solubility tags. We first quantify a highly purified, stable isotope labeling of amino acids in cell culture (SILAC)-labeled version of the solubility tag and use it determine the precise amount of each PrEST by its SILAC ratios. The PrESTs are then spiked into cell lysates and the SILAC ratios of PrEST peptides to peptides from endogenous target proteins yield their cellular quantities. The procedure can readily be multiplexed, as we demonstrate by simultaneously determining the copy number of 40 proteins in HeLa cells. Among the proteins analyzed, the cytoskeletal protein vimentin was found to be most abundant with 20 million copies per cell, while the transcription factor and oncogene FOS only had 6000 copies. Direct quantification of the absolute amount of single proteins is possible via a SILAC experiment in which labeled cell lysate is mixed both with the heavy labeled solubility tag and with the corresponding PrEST. The SILAC-PrEST combination allows accurate and streamlined quantification of the absolute or relative amount of proteins of interest in a wide variety of applications.
Key Points• As little as 5% of kindlin-3 is sufficient to maintain basal platelet and leukocyte functions.• Platelets and neutrophils contain stoichiometric quantities of kindlin-3 and talin-1.Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1. (Blood. 2015;126(24):2592-2600
The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng Mass spectrometry-based proteomics is fast developing in the direction of clinical applications. Therefore, reliable quantification methods for absolute protein concentration determination are indispensible tools for future applications. So far, enzyme-linked immunosorbent assays and similar antibodybased methods excel in the sensitive detection of low levels of proteins in complex matrices, whereas mass spectrometry enables unbiased approaches and can provide unsurpassed specificity. The fact that most proteomes have a very high dynamic range between high and low abundant proteins, in particular for clinical samples, such as plasma and serum, often makes it necessary to use protein depletion of the most abundant proteins (1, 2) and/or elaborate fractionations (3-5) before running the mass spectrometry analysis. This has prompted several investigators to introduce a protein or peptide capture step using specific antibodies to allow for immunoaffinity enrichment prior to the MS analysis. In this way, a "sandwich" assay is obtained, but instead of having a readout in the analysis step based on a second antibody, the analysis step is performed using MS. In such an approach, either the intact protein is captured using an anti-protein antibody (6) or a peptide derived from the protein is captured using an antipeptide antibody that has been raised to the target peptide of interest (7)(8)(9)(10)(11). This is the principle behind stable isotope standards and capture by anti-peptide antibodies (SISCAPA), 1 developed by
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