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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.

SUBMITTER: Heaven MR

PROVIDER: S-EPMC4712743 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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

Heaven Michael R MR Funk Adam J AJ Cobbs Archie L AL Haffey Wendy D WD Norris Jeremy L JL McCullumsmith Robert E RE Greis Kenneth D KD

Journal of mass spectrometry : JMS 20160101 1

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 met ...[more]

PMID: 26757066

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Project description:The ultimate aim of proteomics is to fully identify and quantify the entire complement of proteins and post-translational modifications in biological samples of interest. For the last 15 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition (DDA) mode has been the standard for proteomics when sampling breadth and discovery were the main objectives; multiple reaction monitoring (MRM) LC-MS/MS has been the standard for targeted proteomics when precise quantification, reproducibility, and validation were the main objectives. Recently, improvements in mass spectrometer design and bioinformatics algorithms have resulted in the rediscovery and development of another sampling method: data-independent acquisition (DIA). DIA comprehensively and repeatedly samples every peptide in a protein digest, producing a complex set of mass spectra that is difficult to interpret without external spectral libraries. Currently, DIA approaches the identification breadth of DDA while achieving the reproducible quantification characteristic of MRM or its newest version, parallel reaction monitoring (PRM). In comparative de novo identification and quantification studies in human cell lysates, DIA identified up to 89% of the proteins detected in a comparable DDA experiment while providing reproducible quantification of over 85% of them. DIA analysis aided by spectral libraries derived from prior DIA experiments or auxiliary DDA data produces identification and quantification as reproducible and precise as that achieved by MRM/PRM, except on low?abundance peptides that are obscured by stronger signals. DIA is still a work in progress toward the goal of sensitive, reproducible, and precise quantification without external spectral libraries. New software tools applied to DIA analysis have to deal with deconvolution of complex spectra as well as proper filtering of false positives and false negatives. However, the future outlook is positive, and various researchers are working on novel bioinformatics techniques to address these issues and increase the reproducibility, fidelity, and identification breadth of DIA.

Dataset Information

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

Publications

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

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