An integrated workflow for crosslinking mass spectrometry

Marta L. Mendes, Lutz Fischer, Zhuo A. Chen, Marta Barbon, Francis J. O'Reilly, Sven H. Giese, Michael Bohlke-Schneider, Adam Belsom, Therese Dau, Colin W. Combe, Martin Graham, Markus R. Eisele, Wolfgang Baumeister, Christian Speck, Juri Rappsilber*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

55 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article numbere8994
JournalMolecular Systems Biology
Volume15
Issue number9
DOIs
Publication statusPublished - 1 Sep 2019

Keywords

  • crosslinking mass spectrometry
  • protein–protein interactions
  • proteomics
  • software
  • structural biology

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