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Tyrosine phosphorylation plays a pivotal role in signal transduction, which affects all aspects of cell life. Recently, mass spectrometry (MS) has been a powerful tool for identification of tyrosine phosphorylation. Yet the MS approach is still far from being routine due mainly to low phosphorylation stoichiometry at any given site.
The goal of this work was to identify the phosphotyrosine (pY) residues on a human sorting nexin protein using nanoLC coupled with a triple-quadrupole linear ion-trap mass spectrometer. The initial identification was performed at enhanced survey MS (EMS)-driven information-dependent acquisition (IDA) mode. The subsequent validation and further identification of low-stoichiometry pY sites were carried out using both precursor ion scan-triggered IDA and targeted multiple reaction monitoring (MRM)-triggered IDA. Compared to EMS-IDA analysis, the triple quadrupole–based precursor ion scan and MRM-driven IDA analyses provide significantly increased selectivity and sensitivity for detection of pY residues.
The immunoprecipitation samples from lysates of two transfected cell lines with Src kinase and ACK2 kinase were separated by SDS gel. The bands of interest (1–2 μg) were subjected to tryptic digestion/extraction. The extracts were split into three fractions for nanoLC-MS/MS operated at EMS-IDA, precursor ion scan IDA, and targeted MRM-IDA. The IDA files were used for database searches, and the spectra for identified pY peptides were manually inspected and validated.
The results showed that five pY residues were identified by EMS-IDA and validated by the combined PTM discovery workflow for the Src kinase–catalyzed sample. The estimated stoichiometry of identified pY residues is from 3% to 25%. Furthermore, two pY sites were identified by the PTM discovery approach for the ACK2 kinase–catalyzed sample. The results demonstrate that the triple quadrupole–based precursor ion scan and MRM-driven IDA provide excellent utilities for improved detection of phosphotyrosine residues without additional steps for enrichment of phosphopeptides.