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

Rey, Martial; Dhenin, Jonathan; Kong, Y.; Nouchikian, Lucienne; Filella, Isaac; Duchateau, Magalie; Dupré, Mathieu; Pellarin, Riccardo; Duménil, Gérard; Chamot‐Rooke, Julia

doi:10.1021/acs.analchem.0c04430

Cited by 26 publications

(40 citation statements)

References 54 publications

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“…Samples were flash frozen in liquid nitrogen until further analyses. Protein digestion, cross-linked peptides enrichment, and mass spectrometry analysis were performed as previously described 59 .…”

Section: Methodsmentioning

confidence: 99%

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

et al. 2021

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Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

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Section: Methodsmentioning

confidence: 99%

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

et al. 2021

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“…Thus, protein and peptide-level fractionation is particularly critical to achieve the restraint density required by the use of crosslinks as restraints in integrative structure modeling, especially on low-abundance proteins. While multidimensional fractionation has pushed the limit of tag-free chromatographic enrichment, we envision that new-generation enrichable crosslinkers based on previously underutilized chemistries (Rey et al, 2021;Stadlmeier et al, 2020) will provide opportunities for the selective enrichment of crosslinked peptides.…”

Section: Crosslinking-ms In Biological Discovery: In Situ Structural Interactomicsmentioning

confidence: 99%

Leveraging crosslinking mass spectrometry in structural and cell biology

Graziadei

Rappsilber

2022

Structure

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Crosslinking mass spectrometry (crosslinking-MS) is a versatile tool providing structural insights into protein conformation and protein-protein interactions. Its medium-resolution residue-residue distance restraints have been used to validate protein structures proposed by other methods and have helped derive models of protein complexes by integrative structural biology approaches. The use of crosslinking-MS in integrative approaches is underpinned by progress in estimating error rates in crosslinking-MS data and in combining these data with other information. The flexible and high-throughput nature of crosslinking-MS has allowed it to complement the ongoing resolution revolution in electron microscopy by providing system-wide residue-residue distance restraints, especially for flexible regions or systems. Here, we review how crosslinking-MS information has been leveraged in structural model validation and integrative modeling. Crosslinking-MS has also been a key technology for cell biology studies and structural systems biology where, in conjunction with cryoelectron tomography, it can provide structural and mechanistic insights directly in situ. ll

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“…While proximity labeling chemically 'tags' all proteins in a vicinity, an orthogonal method for protein proximity studies is using chemical crosslinking to covalently connect nearby proteins to each other [25], capturing even weak or transient protein interactions and complexes in a snapshot. Much like proximity labeling, there are a variety of crosslinking reagents available with a range of physiochemical properties, such as reactivity, selectivity, suitability for in vivo work, and different spacer arm length (which dictates the maximum distance between two proximal proteins) and flexibility [26].…”

Section: Crosslinking Proximal Proteins With Mass Spectrometry-cleavable Crosslinkersmentioning

confidence: 99%

Proximity labeling and other novel mass spectrometric approaches for spatiotemporal protein dynamics

Pino

Schilling

2021

Expert Review of Proteomics

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Background: Proteins are highly dynamic and their biological function is controlled by not only temporal abundance changes but also via regulated protein-protein interaction networks, which respond to internal and external perturbations. A wealth of novel analytical reagents and workflows allow studying spatiotemporal protein environments with great granularity while maintaining high throughput and ease of analysis. Areas covered: We review technology advances for measuring protein-protein proximity interactions with an emphasis on proximity labeling, and briefly summarize other spatiotemporal approaches including protein localization, and their dynamic changes over time, specifically in human cells and mammalian tissues. We focus especially on novel technologies and workflows emerging within the past 5 years. This includes enrichment-based techniques (proximity labeling and crosslinking), separationbased techniques (organelle fractionation and size exclusion chromatography), and finally sortingbased techniques (laser capture microdissection and mass spectrometry imaging). Expert opinion: Spatiotemporal proteomics is a key step in assessing biological complexity, understanding refined regulatory mechanisms, and forming protein complexes and networks. Studying protein dynamics across space and time holds promise for gaining deep insights into how protein networks may be perturbed during disease and aging processes, and offer potential avenues for therapeutic interventions, drug discovery, and biomarker development.

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Advanced In Vivo Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions

Cited by 26 publications

References 54 publications

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

Leveraging crosslinking mass spectrometry in structural and cell biology

Proximity labeling and other novel mass spectrometric approaches for spatiotemporal protein dynamics

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