An integrated workflow for crosslinking mass spectrometry

• DOI: 10.15252/msb.20198994
• Publication Date: 2019-09-20T07:55:35Z
• Authors: Marta L Mendes; Lutz Fischer; Zhuo A Chen; Marta Barbon; Francis J O'Reilly; Sven H Giese; Michael Bohlke‐Sc...; Adam Belsom; Therese Dau; Colin W Combe; Martin Graham; Markus R Eisele; Wolfgang Baumeister; Christian Speck; Juri Rappsilber
• Keywords: Models, Molecular; Proteomics; 0301 basic medicine; Medicine (General); Bioinformatics; QH301-705.5; Protein Conformation; 0699 Other Biological Sciences; protein-protein interactions; protein–protein interactions; 0601 Biochemistry and Cell Biology; Mass Spectrometry; 03 medical and health sciences; proteomics; R5-920; Cytosol; Protein Interaction Mapping; Methods; structural biology; Humans; crosslinking mass spectrometry; Biology (General); 0303 health sciences; software; Proteins; 540; Peptide Fragments; 620; K562 Cells; Software

We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software Xi. 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.