Abstract
Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.
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Acknowledgements
We thank R. Spohrer for sample preparation and L. Burton, A. Lau and G. Ivosev for their support with SWATH 2.0. P.N. and S.T. are supported by grants from the Bundesministerium für Bildung und Forschung (BMBF) (Express2Present 0316179C), the Deutsche Forschungsgemeinschaft (DFG) (ST599/1-1, ST599/2-1) and Mainz University (Research Center for Immunotherapy (FZI)). H.L.R. is supported by Swiss National Science Foundation grant P2EZP3_162268. Y.P.-R. is supported by the Biotechnology and Biological Sciences Research Council (BBSRC) 'PROCESS' grant (BB/K01997X/1). A.I.N. is supported by US National Institutes of Health grant 5R01GM94231. R.A. was supported by European Research Council (ERC) AdG 233226 (Proteomics version 3.0) and ERC-2014-AdG 670821 (Proteomicxs 4D), the PhosphonetX project of SystemsX.ch and the Swiss National Science Foundation grant 3100A_166435.
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Contributions
P.N. and S.T. designed and supervised the study; U.D. and L.C.G. prepared the samples and performed the MS measurements; L.C.G., G.R. and H.L.R. executed and supervised the OpenSWATH analyses; P.N. and S.A.T. executed and supervised the SWATH 2.0 analyses; P.N. and B.M. executed and supervised the Skyline analyses; P.N., O.M.B. and L.R. executed and supervised the Spectronaut analyses; C.-C.T. and A.I.N. executed and supervised the DIA-Umpire analyses; J.K., P.N. and Y.P.-R. developed LFQbench; P.N., S.T., J.K., B.M. and O.M.B. performed the benchmark analyses; L.C.G., O.M.B., B.M., H.L.R., S.A.T., C.-C.T., L.R., G.R., A.I.N. and R.A. provided critical input into the project; P.N., J.K. and S.T. wrote the manuscript.
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S.A.T. is employed by SCIEX, and O.M.B. and L.R. are employed by Biognosys AG.
Supplementary information
Supplementary Text and Figures
Supplementary Tables 1–9, Supplementary Figures 1–25 and Supplementary Notes 1–6 (PDF 28116 kb)
Supplementary Code
LFQbench (R-package) source code (ZIP 19693 kb)
Supplementary Data Set 1
List of peptides exclusively identified by DIA-Umpire in the HYE124 64 variable windows experiment (XLSX 131 kb)
Supplementary Data Set 2
Ion library for 32 fixed SWATH windows experiments (ZIP 14442 kb)
Supplementary Data Set 3
Ion library for 32 variable SWATH windows experiments (ZIP 15376 kb)
Supplementary Data Set 4
Ion library for 64 fixed SWATH windows experiments (ZIP 15413 kb)
Supplementary Data Set 5
Ion library for 64 variable SWATH windows experiments (ZIP 14695 kb)
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Navarro, P., Kuharev, J., Gillet, L. et al. A multicenter study benchmarks software tools for label-free proteome quantification. Nat Biotechnol 34, 1130–1136 (2016). https://doi.org/10.1038/nbt.3685
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DOI: https://doi.org/10.1038/nbt.3685
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