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Mol Syst Biol. 2019 Sep;15(9):e8994. doi: 10.15252/msb.20198994.

An integrated workflow for crosslinking mass spectrometry.

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Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
MRC London Institute of Medical Sciences (LMS), London, UK.
DNA Replication Group, Faculty of Medicine, Institute of Clinical Sciences (ICS), Imperial College London, London, UK.
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.


We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.


crosslinking mass spectrometry; protein-protein interactions; proteomics; software; structural biology

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