Accurate precursor mass assignment improves peptide identifica-tion in data-independent acquisition mass spectrometry (DIA-MS)

Proteomics research today no longer simply seeks exhaustive protein identification; increasingly, it is also desirable to obtain robust, large-scale quantitative information. To accomplish this, data-independent acquisition (DIA) has emerged as a promis-ing strategy largely owing to developments in advanced mass spectrometers and sophisticated data analysis methods. Never-theless, 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 search-ing. Compared to conventional spectral library searching, the accuracy and sensitivity of peptide identification were significant-ly increased by incorporating precise precursor masses in DIA data. We demonstrate identification of additional peptides ab-sent from spectral libraries, incl...