Multiplexed MS/MS for improved data-independent acquisition

Journal name:
Nature Methods
Year published:
Published online

In mass spectrometry–based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold.

At a glance


  1. Multiplexed data-independent acquisition with MSX.
    Figure 1: Multiplexed data-independent acquisition with MSX.

    A common implementation of DIA is to use a repeated cycle of wide–isolation window MS/MS scans to cover a mass range. In this example, the 500- to 900-m/z range is covered with 40 scans each sampling a single 10-m/z-wide window. In MSX, each scan isolates five 4-m/z-wide windows before fragment-ion mass analysis. The five windows isolated in each scan are chosen randomly from the set of 100 possible nonoverlapping windows covering the 500- to 900-m/z range. Each mixed MSX spectrum is demultiplexed into the five component spectra corresponding to the isolated windows.

  2. Demultiplexing reduces chemical noise and improves selectivity.
    Figure 2: Demultiplexing reduces chemical noise and improves selectivity.

    (ad) The full b- and y-ion series for the peptide GPLVLEYETYR are plotted from an MSX experiment on the soluble fraction of S. cerevisiae lysate before (a,c) and after (b,d) demultiplexing. Prior to demultiplexing, there are many other peaks of similar or greater intensity than the peak for GPLVLEYETYR (arrow). After demultiplexing, peaks from other precursors are effectively removed, and the true peak is by far the most intense (b). Additionally, the demultiplexed peak (d) contains far less interference than the unprocessed peak (c).

  3. Quantitation of the LVNELTEFAK peptide by MSX and MS1.
    Figure 3: Quantitation of the LVNELTEFAK peptide by MSX and MS1.

    A commercial six-protein digest was spiked into a soluble S. cerevisiae lysate in amounts ranging from 50 attomoles to 100 fmol on-column. MSX data were acquired with an MS1 scan interleaved every ten scans. (a) Signal intensity for each spike-in point (normalized to two background peptides) for the peptide LVNELTEFAK2+ with (right) and without (left) log scaling of the x and y axes. The lower limits of detection were 0.41 fmol and 1.02 fmol for MSX- and MS1-based quantitation, respectively. The slopes of the regression lines were 0.030 ± 0.004 (95% confidence interval) and 0.071 ± 0.002 for MSX and MS1, respectively. (b,c) MS1 (b) and MSX (c) signal at 1.02 fmol. M, M + 1 and M + 2 represent precursor isotopes.


  1. Supplementary Video 1
    Video 1: Supplementary Video 1
    Demonstration of multiplexed data-independent acquisition (MSX) and demultiplexing

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Referenced accessions



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Author information


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

    • Jarrett D Egertson,
    • Gennifer E Merrihew,
    • Brendan X MacLean,
    • Ying S Ting,
    • Donald M Marsh &
    • Michael J MacCoss
  2. Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany.

    • Andreas Kuehn &
    • Markus Kellmann
  3. Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

    • Nicholas W Bateman &
    • Christine C Wu
  4. Thermo Fisher Scientific, San Jose, California, USA.

    • Jesse D Canterbury &
    • Vlad Zabrouskov


A.K., C.C.W., J.D.C., J.D.E., M.J.M., M.K., V.Z. and Y.S.T. designed experiments. A.K., C.C.W., J.D.E., M.J.M., M.K., N.W.B. and V.Z. interpreted results. J.D.E. and M.J.M. wrote the manuscript. A.K. created a firmware modification for the Q-Exactive. A.K. and M.K. provided preliminary data. G.E.M., J.D.E. and N.W.B. performed experiments. B.X.M., D.M.M. and J.D.E. wrote software.

Competing financial interests

All authors received financial support from Thermo Fisher Scientific. A.K., J.D.C., M.K. and V.Z. are employees of Thermo Fisher Scientific, the company that markets and sells the mass spectrometry instrumentation used in this manuscript. Thermo Fisher will make the methods described herein available to their customers in version 2.2 SP1 of the Q-Exactive software.

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Supplementary information


  1. Video 1: Supplementary Video 1 (5,309 KB, Download)
    Demonstration of multiplexed data-independent acquisition (MSX) and demultiplexing

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  1. Supplementary Text and Figures (3.4 MB)

    Supplementary Figures 1–8, Supplementary Tables 1 and 2, Supplementary Data and Supplementary Note

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