Large-scale Proteomics Analysis of the Human Kinome

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis.Reversible protein phosphorylation represents the most common type of post-translational modification (PTM)¹ in eukaryotic organisms. A plethora of studies on a large variety of proteins have established that site-specific phosphorylation events fulfill key functions in the activity control of signaling cascades and networks (1). Cellular protein phosphorylation is controlled by more than 500 members of the protein kinase superfamily, which comprises one of the largest enzyme families encoded by the human genome (2). Protein kinases represent the key elements in phosphorylation-based signal transmission. Aberrant protein kinase expression and/or activity, often because of gene amplification or mutational changes, is involved in pathological processes leading to malignant transformation and tumor development (3). Therefore, protein kinases have emerged as a major class of drug targets for therapeutic intervention (4–6). Given the diversity of molecular mechanisms related to de-regulated kinase function in human cancers, proteomic approaches could significantly enhance our understanding of disease-relevant kinase function and also help to optimize and adjust therapeutic strategies. In addition to assessing protein expression, the analysis of site-specific phosphorylations on protein kinases is of particular relevance, as these PTMs can be indicative of their cellular catalytic activities (7, 8). Protein kinases can not only modulate each other''s functions and activities through site-specific phosphorylation events, but often also undergo site-specific autophosphorylation once they get activated (9). Thus, the comprehensive assessment of kinase-derived phosphopeptides can provide important insights into the regulation of these key players in phosphorylation-controlled signaling.Regulatory enzymes such as protein kinases are often expressed at low cellular levels. This can impede their detection by LC-MS in highly complex peptide mixtures derived from total cell or tissue extracts. These analytical challenges are further aggravated in phosphoproteomic experiments due to the fact that many phosphopeptide species result from sub-stoichiometric phosphorylation events (10). Consequently, phosphopeptide isolation methods have proven to be essential. Among others, techniques such as immobilized metal affinity chromatography or enrichment by means of titanium dioxide (TiO₂)-coated beads have found widespread use in MS-based phosphoproteomics (11–13). In addition, to reduce initial sample complexity, either protein fractionation by gel electrophoresis or peptide separation by strong cation exchange chromatography is typically included in contemporary phosphoproteomics workflows (14–16). These separation techniques in combination with LC-MS on state-of-the-art mass spectrometers enabled the identification of thousands of phosphorylation sites from total cellular extracts (15, 17, 18). Despite these impressive advances, such large-scale efforts require considerable instrument time, and the current methodology is still not comprehensive across the full dynamic range of the entire phosphoproteome. This creates the rationale for sub-proteome analyses to achieve high coverage and analytical sensitivity, which is particularly relevant for members of the protein kinase enzyme family.To date, the only pre-fractionation techniques permitting the enrichment of more than a few protein kinases are affinity capture methods relying on immobilized and kinase-selective small molecule inhibitors (19–21). We and others have demonstrated that combinations of such kinase inhibitor resins efficiently pre-fractionate kinases for subsequent phosphorylation analysis (7, 22, 23). Ideally, capture molecules for kinase proteomics have two properties. First, they should exhibit high non-selectivity within the kinase superfamily. Second, they should efficiently discriminate between protein kinases and other classes of cellular proteins under the biochemical conditions of the pre-fractionation procedure.In our efforts to characterize affinity reagents fulfilling these criteria, we quantitatively compared a selection of immobilized pyrido[2,3-d]pyrimidine-based inhibitors with respect to their proteome-wide kinase binding properties. Based on this assessment, an affinity matrix displaying the small molecule VI16832 was used as an enrichment tool for the comparative expression analysis of protein kinases in different cancer cell lines. The highly efficient VI16832 affinity resin further enabled a large-scale phosphoproteomics survey resulting in the identification and confident assignment of about 1200 phosphorylation sites on more than 200 distinct protein kinases.