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Building high-quality assay libraries for targeted analysis of SWATH MS data
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Building high-quality assay libraries for targeted analysis of SWATH MS data
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Building high-quality assay libraries for targeted analysis of SWATH MS data
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Journal Article
Building high-quality assay libraries for targeted analysis of SWATH MS data
2015
Overview
Targeted proteomics by SRM/MRM or SWATH MS relies on reference assay libraries for peptide identification. This protocol is for building extensive, high-quality reference libraries starting with data acquired by discovery proteomics.
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.
Publisher
Nature Publishing Group UK,Nature Publishing Group
