Proteomics

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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

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BSA.fasta Fasta
MSStudioparameter.xml Xml
NM8013.fasta Fasta
Nm_2h.raw Raw
Nm_4h.raw Raw
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Publications

Advanced <i>In Vivo</i> Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions.

Rey Martial M   Dhenin Jonathan J   Kong Youxin Y   Nouchikian Lucienne L   Filella Isaac I   Duchateau Magalie M   Dupré Mathieu M   Pellarin Riccardo R   Duménil Guillaume G   Chamot-Rooke Julia J  

Analytical chemistry 20210222 9

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 <i>in vivo</i> studies. Pushing in this direction, we developed an advanced <i>in vivo</i> cross-linking mass spectrometry platform to study the cellular interactome of living bacterial cells. It is based on <i>in vivo</i> labeling and involves a one-step  ...[more]

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