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Nat Protoc. 2015 Mar;10(3):426-41. doi: 10.1038/nprot.2015.015. Epub 2015 Feb 12.

Building high-quality assay libraries for targeted analysis of SWATH MS data.

Author information

1
1] Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland. [2] PhD Program in Systems Biology, University of Zurich and ETH Zurich, Zurich, Switzerland.
2
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
3
Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany.
4
1] Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland. [2] SystemsX.ch Biology IT (SyBIT), SystemsX.ch, Zurich, Switzerland.
5
Division of Biomedical Engineering and Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
6
Department of Radiology, Stanford University School of Medicine, Stanford, California, USA.
7
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
8
1] Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland. [2] Faculty of Science, University of Zurich, Zurich, Switzerland.

Abstract

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.

PMID:
25675208
DOI:
10.1038/nprot.2015.015
[Indexed for MEDLINE]

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