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Informatics solutions for SRM assays pose several specific bioinformatics challenges including assay development, generating acquisition methods, and data processing. Furthermore, SRM is often coupled to experimental designs using stable isotope dilution SRM mass spectrometry workflows (SID-SRM-MS) that utilize one or more stable isotope versions of the analyte as internal standards. Skyline, an open-source software suite of tools for targeted proteomics, has emerged as the most widely used platform for SRM-specific assays. Skyline is a freely-available, comprehensive tool with high versatility for SRM assay development and subsequent processing of data acquired on triple quadrupole mass spectrometers. Skyline can be used for peptide and transition selection, assay optimization, retention time scheduling, SRM instrument method export, peak detection/integration, post-acquisition signal processing, and integration with statistical tools and algorithms to generate quantitative results for peptides and proteins. To highlight some of the Skyline SRM functionalities, we describe features including important visual displays and statistical tools, including ‘External Tools’. We discuss Skyline features that are particularly valuable for system suitability assessments, as well as for data sets with posttranslational modifications. Finally, an easy, point-and-click strategy is presented that supports dissemination of SRM data processed in Skyline to the Panorama web data repositories.

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