title: Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics-a prototype design for a single injection assay.
abstract: 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 (13) C 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.
authors.affiliation: Vulcan Analytical, LLC, Birmingham, AL, 35203, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Heaven
authors.affiliation: Department of Psychiatry and Behavioral Neuroscience, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Funk
authors.affiliation: Vulcan Analytical, LLC, Birmingham, AL, 35203, USA.
authors.forename: Archie L
authors.initials: AL
authors.lastname: Cobbs
authors.affiliation: Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
authors.forename: Wendy D
authors.initials: WD
authors.lastname: Haffey
authors.affiliation: Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Norris
authors.affiliation: Department of Psychiatry and Behavioral Neuroscience, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: McCullumsmith
authors.affiliation: Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Greis
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017022:Flow Injection Analysis / Q000379:methods; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D020543:Proteome
keywords: DIA; PAcIFIC; Protalizer; SWATH; label-free quantification