| Reversible protein phosphorylation plays a vital role in the regulation of cellular signaling pathways. With recent breakthrough developments in mass spectrometry-based proteomics technologies, including phosphopeptide enrichment and separation techniques, high-accuracy mass spectrometry and associated bioinformatics, MS-based quantitative phosphoproteomics have now gained great popularity. This technology has enabled the simultaneous identification and quantification of thousands of phosphorylation sites from entire phosphoproteomes.;Mast cells play a central role in type I hypersensitivity reactions and allergic disorders such as anaphylaxis and asthma. In order to understand the molecular architecture underlying mast cell signaling, a systematic, quantitative analysis of the global tyrosine phosphorylation events triggered by activation of the mast cell receptor was performed. We have for the first time substantially characterized and quantified hundreds of tyrosine phosphorylation events in both mouse mast cell line MCP5 cells and mouse bone marrow-derived mast cells, with their temporal phosphorylation profiles providing preliminary insights into the newly discovered phosphorylation sites.;However, this type of analysis does not provide enough information to make precise predictions of the placement of individual phosphorylation events within signaling pathways. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the newly discovered phosphorylation sites. To better understand the molecular mechanism underlying complex cellular signaling networks, we have also developed a hybrid quantitative approach that combines label free and SILAC quantification techniques. Label free quantification is applied to assemble high-density temporal data within a single cell type, either wide-type (WT) or mutant (Mut) cells, providing a list of phosphorylation sites that change in abundance after stimulation of a cellular receptor. SILAC quantification is then used to compare WT and Mut cells across a timecourse of receptor stimulation, providing direct information about how the newly observed phosphorylation sites respond to the mutagenesis. We have successfully applied this approach to ZAP-70 and SLP-76 deficient Jurkat T cell lines. These studies have provided great insights into the essential roles of these proteins in T cell signaling. Many hypotheses have been drawn and follow-up studies could provide directions for future investigation. |
