A simplified in vivo cross-linking mass spectrometry strategy for proteome-wide studies.

ABSTRACT: Chemical cross-linking (XL) coupled to mass spectrometry (MS) has become a powerful approach to probe the structure of protein assemblies. Although most of the applications concerned purified complexes, latest developments focus on large-scale in vivo studies. Pushing in this direction, we describe a new and simplified in vivo XL-MS workflow to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click-chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 1,800 cross-links in a single LC-MS/MS run using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our large-scale approach. Our workflow paves new avenues for the deep in vivo analysis of cellular interactomes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Bacteria Neisseria Meningitidis

SUBMITTER: Martial Rey  

LAB HEAD: Julia Chamot-Rooke

PROVIDER: PXD021553 | Pride | 2021-02-25

REPOSITORIES: Pride