Abstract
AbstractCross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system-wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging to decide for an appropriate analysis workflow. Here, we report a large and flexible synthetic peptide library as reliable instrument to benchmark crosslink workflows. Additionally, we provide a tool, IMP-X-FDR, that calculates the real, experimentally validated, FDR, compares results across search engine platforms and analyses crosslink properties in an automated manner. We apply the library with 6 commonly used linker reagents and analyse the data with 6 established search engines. We thereby show that the correct algorithm and search setting choice is highly important to improve identification rate and reliability. We reach identification rates of up to ~70 % of the theoretical maximum (i.e. 700 unique lysine-lysine cross-links) while maintaining a real false-discovery-rate of <3 % at cross-link level with high reproducibility, representatively showing that our test system delivers valuable and statistically solid results.
Funder
Austrian Science Fund
EC | Horizon 2020 Framework Programme
Vienna Science and Technology Fund
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary
Cited by
14 articles.
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