Data-independent acquisition (DIA) is an emerging mass spectrometry (MS)-based technique for unbiased and reproducible measurement of protein mixtures. DIA tandem mass spectrometry spectra are often highly multiplexed, containing product ions from multiple cofragmenting precursors. Detecting peptides directly from DIA data is therefore challenging; most DIA data analyses require spectral libraries. Here we present PECAN (http://pecan.maccosslab.org), a library-free, peptide-centric tool that robustly and accurately detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, which enables detection of low-abundance analytes with poor precursor ion signal. We demonstrate the chromatographic peak picking accuracy and peptide detection capability of PECAN, and we further validate its detection with data-dependent acquisition and targeted analyses. Lastly, we used PECAN to build a plasma proteome library from DIA data and to query known sequence variants.

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The authors thank L. Käll, A.I. Nesvizhskii, N. Bandeira, and J.K. Eng for insightful discussions. This work was supported by the National Institutes of Health Grants P30 AG013280, R21 CA192983, P41 GM103533, and U54 HG008097. S.H.P. was supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, and Early Career Research Program.

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

    • Ying S Ting
    • , Jarrett D Egertson
    • , James G Bollinger
    • , Brian C Searle
    • , William Stafford Noble
    •  & Michael J MacCoss
  2. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.

    • Samuel H Payne
  3. Department of Computer Science and Engineering, University of Washington, Seattle, Washington, USA.

    • William Stafford Noble


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Y.S.T. and M.J.M. designed the experiments. Y.S.T. developed the algorithms with input from J.D.E., S.H.P., B.C.S., W.S.N., and M.J.M. Y.S.T. performed the analyses. Y.S.T. and J.G.B. acquired the data. Software was written by Y.S.T. with input from J.D.E. and B.C.S. The manuscript was written by Y.S.T. with substantial input from J.D.E., S.H.P., W.S.N., and M.J.M.

Competing interests

The MacCoss Lab at the University of Washington has a sponsored research agreement with Thermo Fisher Scientific, the manufacturer of the instrumentation used in this research. Additionally, M.J.M. is a paid consultant for Thermo Fisher Scientific.

Corresponding author

Correspondence to Michael J MacCoss.

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