This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Epidermal growth factor receptor (EGFR) tyrosine kinase plays an important role in regulating cell growth, proliferation, and migration. Differential phosphorylation of specific tyrosine residues in EGFR in response to diverse external stimuli (e.g., exposure to EGF, ATP, cell injury, etc.) serves as the key link between these stimuli and the internal signaling pathways that they activate. EGFR phosphosite-specific antibodies have been used as highly sensitive tools to monitor phosphorylation site occupancy, however, they suffer from a lack of specificity. Recently, highly sensitive mass spectrometric-based detection strategies have been described to characterize the EGFR tyrosine phosphorylation cascade under a variety of conditions. We are exploring the optimization of sample preparation, phosphopeptide enrichment and detection strategies for the study of EGFR phosphopeptides. Human epidermoid carcinoma A431 cells, which over-express EGFR, or the porcine aortic endothelial (PAE) cell line transfected with EGFR expression constructs, or mouse corneal epithelial cells, were grown in culture to confluence, harvested in the presence of a cocktail of protease and phosphatase inhibitors, and EGFR was subjected to immunoprecipitation. Eluted EGFR, or control, commercially-available purified EGFR (derived from A431 cells) was subjected to SDS-PAGE and in-gel digestion with a panel of proteases. Peptides were eluted and subjected to enrichment by reversed-phase, ion exchange, metal ion affinity, or titanium dioxide affinity chromatography. Phosphopeptides were analyzed by MALDI-TOF MS using a variety of matrices and matrix additives in the positive and negative ion modes. We have recovered EGFR from cells in culture with high yield and purity using immunoprecipitation followed by SDS-PAGE. We have explored the use of multiple proteases for in-gel digestion, to target, in particular large tyrosine-containing tryptic peptides that may have been unrepresented in previous MS analyses. Using optimized procedures for recovery from gel, we have subjected the EGFR peptides to various forms of chromatographic enrichment and separation techniques to exploit the differential binding and elution of EGFR phosphopeptides. Using an optimized phosphopeptide recovery and enrichment protocol, followed by MALDI-TOF MS peptide mapping with a Bruker Reflex IV and nanoLC/MSn analysis with a Waters Acuity NanoLC interfaced to an Advion Nanomate and Thermo-Fisher LTQ-Orbitrap MS, we have been able to establish the differential phosphorylation of EGFR in response to injury, and to UTP or EGF stimulation. Phosphorylation of EGFR following UTP stimulation attenuates rapidly compared to direct stimulation of E1PAE cells by EGF. The intensity of EGFR phosphorylation was found to be significantly reduced when cells were stimulated by UTP, as opposed to stimulation by EGF. We observed that Grb2 binding gradually increases over time and reaches its peak at 45 min post-EGF stimulation and attenuates rapidly thereafter. Mutation of tyrosine residues 1068 and 1086 to phenylalanine reduced Grb2 binding when cells were stimulated with UTP, but a smaller decrease was observed when stimulated with EGF. We have also found that neither injury nor UTP, but EGF recruits Grb2 to EGFR. Our data indicates that this is due to the differential phosphorylation of EGFR tyrosine residues that results from stimulation with different agonists.