Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions

Hugo Gizardin-Fredon, Paulo E. Santo, Marie-Eve Chagot, Bruno Charpentier, Tiago M. Bandeiras, Xavier Manival, Oscar Hernandez-Alba and Sarah Cianférani ()
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Hugo Gizardin-Fredon: CNRS
Paulo E. Santo: Apartado 12, 2781-901
Marie-Eve Chagot: Université de Lorraine
Bruno Charpentier: Université de Lorraine
Tiago M. Bandeiras: Apartado 12, 2781-901
Xavier Manival: Université de Lorraine
Oscar Hernandez-Alba: CNRS
Sarah Cianférani: CNRS

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: Abstract 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.

Date: 2024
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DOI: 10.1038/s41467-024-47732-4

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