Advanced <i>In Vivo</i> Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions
Martial Rey, Jonathan Dhenin, Youxin Kong, Lucienne Nouchikian, Isaac Filella, Magalie Duchateau, Mathieu Dupré, Riccardo Pellarin, Guillaume Duménil, Julia Chamot‐Rooke
Abstract
High Resolution Image Download MS PowerPoint Slide 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).