Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level - DSA-IPHC
Preprints, Working Papers, ... (Preprint) Year : 2023

Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level

Abstract

Mass photometry (MP) is a versatile, fast and low sample-consuming biophysical technique that gained interest in structural biology to study noncovalent assemblies in native conditions. We report here on a novel method to perform MP analysis in denaturing conditions (dMP) and its application for fast, accurate and straightforward optimization of chemical reactions in cross-linking mass spectrometry (XL-MS) workflows. dMP consists in a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. The proposed single-molecule method clearly overcomes the limitations and outperforms gold standard SDS-PAGE, as illustrated on several biological complexes. dMP provides an unprecedented and unmatched in-solution quantification of all coexisting XL species, including sub-complexes and non-specific XL aggregates, along with identification of significantly higher numbers of XL dipeptides in MS. We anticipate single-molecule dMP to be a high-impact game-changer for the XL-MS community with the potential to leverage the quality and reliability of XL-MS datasets.
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Dates and versions

hal-04535064 , version 1 (05-04-2024)

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  • HAL Id : hal-04535064 , version 1

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Hugo Gizardin-Fredon, Paulo E Santo, Marie-Eve Chagot, Bruno Charpentier, Tiago M Bandeiras, et al.. Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level. 2024. ⟨hal-04535064⟩
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