An integrated workflow for crosslinking mass spectrometry

• Published in: Molecular systems biology Vol. 15; no. 9; pp. e8994 - n/a
• Main Authors: Mendes, Marta L, Fischer, Lutz, Chen, Zhuo A, Barbon, Marta, O'Reilly, Francis J, Giese, Sven H, Bohlke‐Schneider, Michael, Belsom, Adam, Dau, Therese, Combe, Colin W, Graham, Martin, Eisele, Markus R, Baumeister, Wolfgang, Speck, Christian, Rappsilber, Juri
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.09.2019; EMBO Press; John Wiley and Sons Inc; Springer Nature
• Subjects: Crosslinking; crosslinking mass spectrometry; Cytosol - chemistry; Data analysis; Datasets; Digestion; EMBO22; EMBO56; Humans; K562 Cells; Laboratories; Lysates; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Method; Methods; Models, Molecular; Peptide Fragments - chemistry; Peptides; Protein Conformation; Protein Interaction Mapping - methods; Proteins; Proteins - chemistry; protein–protein interactions; proteomics; Proteomics - methods; Quantitative analysis; Scholarships & fellowships; Scientific imaging; Software; structural biology; Tryptic peptides; Workflow; United Kingdom > UK; protein–protein interactions; crosslinking mass spectrometry; structural biology; proteomics; software; protein-protein interactions
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.20198994

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. Synopsis A new workflow combining sequential digestion and the search software Xi increases the number of identified crosslinks in a wide range of applications. By detecting dynamic protein interactions, crosslinking is synergistic with other structural approaches and is promising for drug development. Sequential digestion outperforms parallel digestion by shortening long peptides and thus facilitates crosslink identification. xiSEARCH outperforms other search algorithms. A dynamic interaction identified in the OCCM complex, and not seen before by cryoEM, is a potential target for cancer therapy. Graphical Abstract A new workflow combining sequential digestion and the search software Xi increases the number of identified crosslinks in a wide range of applications. By detecting dynamic protein interactions, crosslinking is synergistic with other structural approaches and is promising for drug development.