Ultradeep Phosphoproteomics for Assessing Protein Kinase Inhibitor Selectivity on a Proteome Scale

beads were analyzed using data-independent acquisition (DIA), while tyrosine phosphopeptides enriched with SH2-Superbinder were analyzed via data-dependent acquisition (DDA). The comprehensive phosphoproteomic analysis identified that 97 and 316 phosphosites were significantly altered upon Zanubrutinib stimulation in the DDA and DIA data sets, respectively. Bioinformatics analysis of these phosphoproteins provided a detailed selectivity profile of Zanubrutinib, offering insights into its mechanism of action at the molecular level. Compared to existing methods, our approach is more comprehensive, has higher throughput, and is more precise─not only for PKI selectivity assessment but also for broader cell signaling research.