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Multiplexed MS/MS for improved data-independent acquisition


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.

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Figure 1: Multiplexed data-independent acquisition with MSX.
Figure 2: Demultiplexing reduces chemical noise and improves selectivity.
Figure 3: Quantitation of the LVNELTEFAK peptide by MSX and MS1.

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The authors acknowledge financial support from US National Institutes of Health grants R01 GM103551, P41 GM103533 and F31 AG037265.

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Authors and Affiliations



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.

Corresponding author

Correspondence to Michael J MacCoss.

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Competing 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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1 and 2, Supplementary Data and Supplementary Note (PDF 3353 kb)

Supplementary Video 1

Demonstration of multiplexed data-independent acquisition (MSX) and demultiplexing (MOV 5308 kb)

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Egertson, J., Kuehn, A., Merrihew, G. et al. Multiplexed MS/MS for improved data-independent acquisition. Nat Methods 10, 744–746 (2013).

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