Fig. 1.

Detection and quantification of phosphorylation at EGFR Ser⁹⁹¹ by SRM. A, trypsin-digested immunopurified EGFR was analyzed by high resolution LC-MS/MS (Proxeon LC/source; Thermo LTQ-Orbitrap). The MS/MS spectrum of the EGFR peptide MHLPpS⁹⁹¹PTDSNFYR (where pS is phosphoserine) is shown (upper panel). The y and b series ions are indicated. Arrows point to product ions corresponding to parent − 98 and y10 −98 ions (as indicated), which reflect the neutral loss of phosphoric acid from the Ser(P) side chain. B, SRM LC elution profiles plotting the summed signal intensities of ions corresponding to singly phosphorylated MHLPSPTDSNFYR (precursor, m/z = 822.85, z = 2) transitioning to 1) precursor − 98/2 (m/z = 773.86, z = 2) and 2) y10 − 98 (m/z = 1165.50, z = 1) at various time points after EGF stimulation as indicated. C, histogram depicting the integrated SRM signal intensities normalized to a non-phosphorylated EGFR peptide in the same sample. Error bars indicate S.D. from three biological repeats of the experiment. D and E, Western blot (WB) analysis of whole-cell protein extracts from HEK-EGFR cells harvested following EGF treatment for the indicated durations. Antibodies specific to the EGFR phosphoepitope containing Tyr(P)¹⁰⁹² (D) or that recognize the FLAG epitope on EGFR-FLAG protein (E) were used to probe the filters, which were further developed with appropriate secondary antibody-enzyme conjugates and chemiluminescence imaging (see “Experimental Procedures”). pY, phosphotyrosine.