Developing tyrosine phosphoproteome libraries and dual quantification using a hybrid DIA approach

Abstract

Protein tyrosine phosphorylation plays a critical role in initiating upstream cellular signaling transduction. However, the challenge in biological samples is the variability in relative concentrations (0.1%) of site-specific tyrosine phosphorylation on proteins. To navigate these fluctuations and accurately quantify the absolute levels of tyrosine phosphosites among different samples, we reported a hybrid data-independent acquisition-parallel reaction monitoring (DIA-PRM) MS technique for the robust identification and quantification of the phosphoproteome, the establishment of a comprehensive library of tyrosine phosphosites, and the specific assessment of changes in tyrosine phosphorylation. In our model study on non-small cell lung cancer cells, our PRM strategy accomplished by a spiked-in synthetic heavy phosphopeptide demonstrated reliable targeted quantification of the pY1197 on EGFR, revealing levels of 2.5, 4.9, and 5.3 fmol in pervanadate (PV)-treated cells at 0, 15, and 30 min, respectively. Additionally, DIA-extensive phosphoproteomic analysis provided 2765 tyrosine phosphosites within 14,961 global phosphosites corresponding to 1536 phosphoproteins, contributing to the phospho-library establishment and relative quantification of phosphorylation level, especially in the PV-treated time-dependent increase of ErbB signaling pathway. This hybrid DIA-PRM approach will advance the application of precise measurement of changes in multiple phosphotyrosine residues and enhance our understanding of phosphoproteomic dynamics in drug-resistant cascades.