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

Abstract

AbstractProtein 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.