DIA data analysis with unambiguously determined precursor mass

Proteomics research today no longer simply seeks exhaustive protein identification; now, robust, large-scale quantitative information is sought. For this purpose, data-independent acquisition (DIA) has emerged as a promising strategy largely owing to the developments in advanced mass spectrometers and sophisticated data analysis methods. However, the highly complex multiplexed MS/MS spectra produced by DIA remain challenging to interpret. Here, we present a novel strategy to analyze DIA data, based on unambiguous precursor mass assignment through the mPE-MMR (multiplexed post-experimental monoisotopic mass refinement) procedure, and combined with complementary multi-stage database searching. Compared to conventional spectral library searching, the accuracy and sensitivity of peptide identification were effectively increased by assigning precise precursor masses to DIA data. Additional peptides absent from spectral libraries, including sample-specific mutated peptides and post-translationally-modified peptides, were identified by MS-GF+ and MODa/MODi multi-stage database searching with the precursor masses determined. This first use of unambiguously-determined precursor masses to mine DIA data demonstrates considerable potential for further exploitation of this rich experimental data.