Fiche publication
Date publication
avril 2024
Journal
Nature communications
Auteurs
Membres identifiés du Cancéropôle Est :
Pr CHARPENTIER Bruno
,
Dr CIANFERANI Sarah
,
Dr MANIVAL Xavier
Tous les auteurs :
Gizardin-Fredon H, Santo PE, Chagot ME, Charpentier B, Bandeiras TM, Manival X, Hernandez-Alba O, Cianférani S
Lien Pubmed
Résumé
Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.
Mots clés
Cross-Linking Reagents, chemistry, Protein Denaturation, Photometry, methods, Proteins, chemistry, Electrophoresis, Polyacrylamide Gel, methods, Protein Interaction Mapping, methods, Mass Spectrometry, methods, Humans
Référence
Nat Commun. 2024 04 25;15(1):3516
