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Multiplexed peptide analysis using data-independent acquisition and Skyline

Nature Protocols volume 10, pages 887–903 (2015)Cite this article

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Abstract

Here we describe the use of data-independent acquisition (DIA) on a Q-Exactive mass spectrometer for the detection and quantification of peptides in complex mixtures using the Skyline Targeted Proteomics Environment (freely available online at http://skyline.maccosslab.org). The systematic acquisition of mass spectrometry (MS) or tandem MS (MS/MS) spectra by DIA is in contrast to DDA, in which the acquired MS/MS spectra are only suitable for the identification of a stochastically sampled set of peptides. Similarly to selected reaction monitoring (SRM), peptides can be quantified from DIA data using targeted chromatogram extraction. Unlike SRM, data acquisition is not constrained to a predetermined set of target peptides. In this protocol, a spectral library is generated using data-dependent acquisition (DDA), and chromatograms are extracted from the DIA data for all peptides in the library. As in SRM, quantification using DIA data is based on the area under the curve of extracted MS/MS chromatograms. In addition, a quality control (QC) method suitable for DIA based on targeted MS/MS acquisition is detailed. Not including time spent acquiring data, and time for database searching, the procedure takes ∼1–2 h to complete. Typically, data acquisition requires roughly 1–4 h per sample, and a database search will take 0.5–2 h to complete.

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Figure 1: MS/MS analysis in data-dependent acquisition and data-independent acquisition.
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Figure 9: Skyline: QC peptide GILFVGSGVSGGEEGAR++.
Figure 10: Skyline DIA data: LGEHNIDVLEGNEQFINAAK+++.
Figure 11
Figure 12: Skyline: QC peptide LTILEELR++.
Figure 13: Skyline DIA data: NYIIEELNVR++.
Figure 14: Skyline DIA data: TYAAEIAHNISAK.
Figure 15: Skyline DIA data: VSLDDLQQSIEEDEDHVQST.

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Acknowledgements

Financial support for this work was provided from US National Institutes of Health grants P41 GM103533, R01 GM103551, R21 CA192983 and U54 HG008097.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Jarrett D Egertson, Brendan MacLean, Richard Johnson & Michael J MacCoss

  2. Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany

    Yue Xuan

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  1. Jarrett D Egertson
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Contributions

J.D.E., B.M., R.J., Y.X. and M.J.M. developed and optimized the protocol. J.D.E. drafted the text of the manuscript. R.J. prepared samples and acquired data presented in ANTICIPATED RESULTS section.

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Correspondence to Michael J MacCoss.

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Competing interests

M.J.M. is a paid consultant for Thermo Fisher Scientific. Y.X. is an employee of Thermo Fisher Scientific. The MacCoss laboratory receives research support from Thermo Fisher Scientific.

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Egertson, J., MacLean, B., Johnson, R. et al. Multiplexed peptide analysis using data-independent acquisition and Skyline. Nat Protoc 10, 887–903 (2015). https://doi.org/10.1038/nprot.2015.055

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