Combining chemical cross-linking and mass spectrometry of intact protein complexes to study the architecture of multi-subunit protein assemblies

Caroline Haupt*, Tommy Hofmann, Sabine Wittig, Susann Kostmann, Argyris Politis, Carla Schmidt

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)
197 Downloads (Pure)

Abstract

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement. In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

Original languageEnglish
Article numbere56747
Number of pages12
JournalJournal of Visualized Experiments
VolumeNovember 2017
Issue number129
Early online date28 Nov 2017
DOIs
Publication statusPublished - Nov 2017

Keywords

  • Biochemistry
  • Cross-linking
  • Data analysis
  • Issue 129
  • Mass spectrometry
  • Native mass spectrometry
  • Protein complexes
  • Protein interactions
  • Protein network
  • Protein stoichiometry

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