Analysis of Protein Tyrosine Phosphorylation by Nanoelectrospray Ionization High-Resolution Tandem Mass Spectrometry and Tyrosine-Targeted Product Ion Scanning

A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified.