Systematic evaluation of data‐independent acquisition for sensitive and reproducible proteomics‐a prototype design for a single injection assay

Data‐independent acquisition (DIA)‐based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass‐isolation windows. Our findings indicate that the most sensitive single injection LC‐DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment‐ions (SWATH) approach with 26 m/z isolation windows across the entire 400–1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co‐eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal‐to‐noise of peptide fragment‐ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide ¹³C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long‐standing aim of quantitative proteomics, and is a so‐called trait of DIA, we sought to determine whether precursor‐level chromatograms used in some methods rather than their fragment‐level counterparts have similar precision. Our data show that extracted fragment‐ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.