Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understanding molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multistep enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder toward pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds compared to the two-step method. Specifically, we were able to identify 511 nonredundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson's correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyr-dependent signaling network controlling cellular behaviors such as drug resistance.
Keywords: LC−MS/MS; SH2 superbinder; phosphopeptide enrichment; phosphoproteomics; protein tyrosine phosphorylation.