Advanced In Vivo Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions
Open Access
- 22 February 2021
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 93 (9), 4166-4174
- https://doi.org/10.1021/acs.analchem.0c04430
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 developed an advanced in vivo cross-linking mass spectrometry platform 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 3300 cross-links for the LC-MS/MS analysis of a biological triplicate 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 proteome-wide approach. Our workflow paves new avenues for the large-scale and nonbiased analysis of protein–protein interactions. All raw data, databases, and processing parameters are available on ProteomeXchange via PRIDE repository (data set identifier PXD021553).Funding Information
- Agence Nationale de la Recherche (BIP:BIP, ANR-10-BINF-03-13, CLICKMASSLINK-ANR 09-PIRI-0006, T4PNanoAction ANR-18-CE11-0022)
- H2020 European Institute of Innovation and Technology (EPIC-XS : 823839)
- Centre National de la Recherche Scientifique
- Institut Pasteur
This publication has 54 references indexed in Scilit:
- Cleavable Cross-Linkers and Mass Spectrometry for the Ultimate Task of Profiling Protein–Protein Interaction Networks in VivoJournal of Proteome Research, 2020
- In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human MitochondriaJournal of Proteome Research, 2019
- Development of Large-scale Cross-linking Mass SpectrometryMolecular & Cellular Proteomics, 2018
- In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide LevelJournal of Proteome Research, 2017
- Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC)Journal of Proteome Research, 2017
- Proteome-wide profiling of protein assemblies by cross-linking mass spectrometryNature Methods, 2015
- A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living CellsMolecular & Cellular Proteomics, 2014
- Novel Amidinating Cross-Linker for Facilitating Analyses of Protein Structures and InteractionsAnalytical Chemistry, 2010
- Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometryThe EMBO Journal, 2010
- Identification of cross-linked peptides from large sequence databasesNature Methods, 2008