Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows

Manuel Matzinger (), Adrian Vasiu, Mathias Madalinski, Fränze Müller, Florian Stanek and Karl Mechtler ()
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Manuel Matzinger: Vienna BioCenter (VBC)
Adrian Vasiu: Vienna BioCenter (VBC)
Mathias Madalinski: Vienna BioCenter (VBC)
Fränze Müller: Vienna BioCenter (VBC)
Florian Stanek: Vienna BioCenter (VBC)
Karl Mechtler: Vienna BioCenter (VBC)

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: Abstract Cross-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

Date: 2022
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DOI: 10.1038/s41467-022-31701-w

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