Multiplexed MS/MS for improved data-independent acquisition

A multiplexing strategy for data-independent acquisition (DIA)-based mass spectrometry addresses the limitation of low precursor selectivity to make DIA more practical for peptide analysis. In mass spectrometry–based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold.