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.
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
Nat Protoc. 2015 Mar;10(3):426-41. doi: 10.1038/nprot.2015.015. Epub 2015 Feb 12.
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.
靶向蛋白质组学通过选择/多重反应监测（S/MRM），或者在更大的规模上通过 SWATH（所有理论谱图的顺序窗口采集）MS（质谱），通常依赖于肽段鉴定的谱参考文库。因此，这些文库的质量和覆盖范围对于方法的性能至关重要。在这里，我们提供了一个详细的方案，该方案已成功用于构建高质量、广泛的参考文库，支持通过 SWATH MS 进行靶向蛋白质组学。我们描述了该过程的每一步，包括通过发现蛋白质组学进行的数据采集、肽谱匹配（PSM）的断言、共识谱的生成以及唯一定义每个靶向肽的 MS 坐标的编译。详细介绍了假发现率（FDR）控制、保留时间归一化和翻译后修饰肽处理等关键步骤。最后，我们展示了如何使用该文库使用开源软件 Skyline 提取 SWATH 数据。该方案需要 2-3 天才能完成，具体取决于文库的范围和可用的计算资源。
