Two NSCLC cell lysate of PC9 and CL68 were processed by both DDA and DIA mode including reverse-phase (RP) fractionation. The DDA data were searched by MaxQuant, whereas DIA was analyzed by Spectronaut in both library-based and direct DIA mode. a Summary of phosphopeptides identified by single-shot DDA, StageTip fractionated DDA (7 fractions run in duplicate), library-based DIA (libDIA), and direct DIA (dirDIA). b Phosphosite identification comparison. The single-shot DDA and DIA were acquired in triplicate. c Overlap of phosphosites between dirDIA and libDIA. d Distribution coefficient of variation, CV% of phosphosites of PC9 cell (n = 9,665 for DDA, 19,007 for dirDIA, and 30,260 for libDIA. A median coefficient of variation (CV) value of 13.0%, 4.3%, and 5.2% were obtained for DDA, dirDIA, and libDIA in PC9, respectively. e Distribution of phosphosite abundance rank of commonly quantified 9456 phosphosites in DDA, DDA match between runs (represented as DDA*), libDIA, and dirDIA. f Phosphosites identification per each abundance group across triplicate measurements. The blue, yellow, and red lines represent sites quantified in all the three, two, or only in one replicate, respectively. g Missing values across different abundance groups. Source data are provided as a Source Data file.