Proteomics pp 223-233 | Cite as

Generation of High-Quality SWATH® Acquisition Data for Label-free Quantitative Proteomics Studies Using TripleTOF® Mass Spectrometers

  • Birgit Schilling
  • Bradford W. Gibson
  • Christie L. HunterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)


Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data.

Key words

Mass spectrometry SWATH acquisition Data-independent acquisitions Variable windows Quantitation Proteomics 



We acknowledge support from the NIH shared instrumentation grant for the TripleTOF 6600 system at the Buck Institute (1S10 OD016281, B.W.G.).


  1. 1.
    Collins BC, Gillet LC, Rosenberger G et al (2013) Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system. Nat Methods 10:1246–1253CrossRefPubMedGoogle Scholar
  2. 2.
    Gillet LC, Navarro P, Tate S et al (2012) Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics 11(O111):016717PubMedGoogle Scholar
  3. 3.
    Liu Y, Buil A, Collins BC et al (2015) Quantitative variability of 342 plasma proteins in a human twin population. Mol Syst Biol 11:786CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Selevsek N, Chang CY, Gillet LC et al (2015) Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH-mass spectrometry. Mol Cell Proteomics 14:739–749CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Egertson JD, MacLean B, Johnson R et al (2015) Multiplexed peptide analysis using data-independent acquisition and Skyline. Nat Protoc 10:887–903CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hunter CL, Collins B, Gillet L, et al. (2014) Increasing depth of coverage in data independent acquisition with acquisition improvements and higher sample loads. Proccedings of the 61st Annual ASMS Conference on Mass Spectrometry & Allied Topics, Baltimore, MD, June 15–19, 2014Google Scholar
  7. 7.
    Rost HL, Rosenberger G, Navarro P et al (2014) OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data. Nat Biotechnol 32:219–223CrossRefPubMedGoogle Scholar
  8. 8.
    MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966–968CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lambert JP, Ivosev G, Couzens AL et al (2013) Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition. Nat Methods 10:1239–1245CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Bruderer R, Bernhardt OM, Gandhi T et al (2015) Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Mol Cell Proteomics 14:1400–1410CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wang J, Tucholska M, Knight JD et al (2015) MSPLIT-DIA: sensitive peptide identification for data-independent acquisition. Nat Methods 12(12):1106–1108, OnlineCrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Tsou CC, Avtonomov D, Larsen B et al (2015) DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics. Nat Methods 12:258–264, 7 p following 264CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Li Y, Zhong CQ, Xu X et al (2015) Group-DIA: analyzing multiple data-independent acquisition mass spectrometry data files. Nat Methods 12(12):1105–1106, OnlineCrossRefPubMedGoogle Scholar
  14. 14.
    Rardin MJ, Schilling B, Cheng LY et al (2015) MS1 peptide ion intensity chromatograms in MS2 (SWATH) data independent acquisitions. improving post acquisition analysis of proteomic experiments. Mol Cell Proteomics 14:2405–2419CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhang Y, Bilbao A, Bruderer T et al (2015) The use of variable Q1 isolation windows improves selectivity in LC-SWATH-MS acquisition. J Proteome Res 14:4359–4371CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Birgit Schilling
    • 1
  • Bradford W. Gibson
    • 1
  • Christie L. Hunter
    • 2
    Email author
  1. 1.The Buck Institute for Research on AgingRedwood CityUSA
  2. 2.SCIEXFraminghamUSA

Personalised recommendations