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

Hugo Gizardin-Fredon (1) , Paulo E Santo (2) , Marie-Eve Chagot (3) , Bruno Charpentier (3) , Tiago M Bandeiras (2) , Xavier MANIVAL (3) , Oscar Hernandez-Alba (1) , Sarah Cianférani (1)

1 LSMBO - Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg]
2 IBET - Instituto de Biologia Experimental e Tecnológica
3 IMoPA - Ingénierie Moléculaire et Physiopathologie Articulaire

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.

Domaines: Chimie; Sciences du Vivant [q-bio]

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⟩