Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system. Molecular & Cellular Proteomics 1:376 -386, 2002.Proteomics, the large scale study of the protein complement of a cell or tissue, has its origins in the technology of two-dimensional (2D) 1 gel electrophoresis invented more than 25 years ago (1, 2). In 2D gel electrophoresis, quantitation is achieved by recording differences in the staining pattern of proteins derived from two states of cell populations or tissues. Therefore, in addition to obtaining increasingly higher resolution, technology improvements in the 2D gel community have been directed toward the image analysis of 2D gels and the relative quantitation of protein spots by their intensity of staining (3-6). Mass spectrometry has long been used in a quantitative manner in the small molecule field (7). Pharmacological researchers, for example, use isotopically labeled analogs of the compound of interest and add a known amount to the sample for analysis. This is because mass spectrometry is not quantitative per se because of varying detector response, differential ionization yields for different substances, and other factors. Observed peak ratios for isotopic analogs, however, are highly accurate, because there are no chemical differences between the species, and they are analyzed in the same experiment.One of the first uses of isotopic labels in proteomics was for improved sequence assignment in peptide sequencing by tandem mass spectrometry b...

Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

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To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

Closely related signals often lead to very different cellular outcomes. We found that the differentiation of human mesenchymal stem cells into bone-forming cells is stimulated by epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF). We used mass spectrometry-based proteomics to comprehensively compare proteins that were tyrosine phosphorylated in response to EGF and PDGF and their associated partners. More than 90% of these signaling proteins were used by both ligands, whereas the phosphatidylinositol 3-kinase (PI3K) pathway was exclusively activated by PDGF, implicating it as a possible control point. Indeed, chemical inhibition of PI3K in PDGF-stimulated cells removed the differential effect of the two growth factors, bestowing full differentiation effect onto PDGF. Thus, quantitative proteomics can directly compare entire signaling networks and discover critical differences capable of changing cell fate.

To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.