Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS. We describe each step of the process, including data acquisition by discovery proteomics, assertion of peptide-spectrum matches (PSMs), generation of consensus spectra and compilation of MS coordinates that uniquely define each targeted peptide. Crucial steps such as false discovery rate (FDR) control, retention time normalization and handling of post-translationally modified peptides are detailed. Finally, we show how to use the library to extract SWATH data with the open-source software Skyline. The protocol takes 2–3 d to complete, depending on the extent of the library and the computational resources available.
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We thank C. Ludwig and S. Bader for their discussions and feedback on the manuscript, L. Blum for implementation of iProphet support in MAYU, H. Röst for packaging msproteomicstools, J. Slagel for including the qtofpeakpicker and the new MAYU version in the TPP, and the PRIDE Team for maintaining the ProteomeXchange platform. This work has been financially supported by the Framework Programme 7 of the European Commission through SysteMTb (241587), UNICELLSYS (201142), PRIME-XS (262067) and ProteomeXchange (260558), a European Research Council advanced grant Proteomics v3.0 (233226), the Federal Ministry of Education and Research (e:Bio Express2Present, 0316179C) and the Forschungszentrum Immunologie of the University Medical Center Mainz.
R.A. holds shares of Biognosys AG, which operates in the field covered by the article (products are Spectronaut software and iRT-kit).
Integrated supplementary information
(a) Violin plots show the intrinsic fragment ion spectrum variability of three yeast sample injections converted with three DDA centroiding algorithms.
(b) Violin plots show how well the relative intensities of the six most intense fragment ions obtained by a certain centroiding algorithm compare to the relative intensities of the same six fragments in SWATH MS data.
The spectrum similarity was determined using the normalised spectral contrast angle as described by Toprak and co-workers41. N indicates the number of pairwise comparisons of fragment ion spectra for the same peptide precursors between each data file and S indicates the estimated level of dissimilarity obtained by comparing the experimental score distribution to the score distribution of a perturbation benchmark dataset (the lower S, the more similar the spectra).
(c) Depending on the centroiding algorithm implemented in the different peak pickers, the number of peptide and protein identifications from a database search vary slightly (after filtering for protein-level FDR=1%). The size of the circles is proportional to the number of identifications.
The software MAYU estimates the FDRs on PSM (mFDR), peptide (pepFDR), and protein (protFDR) level with respect to the applied iProphet probability threshold. The data is based on the case study described in the protocol, which consists of three whole cell lysates of yeast. As comparison the protein FDR of a very large data set of 331 runs of different human cells and tissues is shown (Pan Human Library as described by Rosenberger and colleagues23).
a) Violin plots show the intrinsic fragment ion spectrum variability of three injections of a yeast sample converted with the qtofpeakpicker and summarised using either the best replicate fragment ion spectrum or consensus algorithm implemented in SpectraST11.
(b) Violin plots show how well the relative intensities of the six most intense fragment ions compare to the relative intensities of the same six fragments in SWATH MS data.
The spectrum similarity was determined using the normalised spectral contrast angle as described by Toprak and co-workers41. N indicates the number of pairwise comparisons of fragment ion spectra for the same peptide precursors between each data file and S indicates the estimated level of dissimilarity obtained by comparing the experimental score distribution to the score distribution of a perturbation benchmark dataset (the lower S, the more similar the spectra). Common neutral losses were included in the comparison.
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Schubert, O., Gillet, L., Collins, B. et al. Building high-quality assay libraries for targeted analysis of SWATH MS data. Nat Protoc 10, 426–441 (2015). https://doi.org/10.1038/nprot.2015.015
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