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

Gizardin-Fredon, H.; Santo, P. E.; Chagot, M.-E.; Charpentier, B.; Bandeiras, T. M.; Manival, X.; Hernandez-Alba, O.; Cianferani, S.

doi:10.1101/2023.05.30.542861

bioRxiv · 10.1101/2023.05.30.542861

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

Gizardin-Fredon, H.·Santo, P. E.·Chagot, M.-E.·Charpentier, B.·Bandeiras, T. M.·Manival, X.·Hernandez-Alba, O.·Cianferani, S.

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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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Gizardin-Fredon, H., Santo, P. E., Chagot, M.-E., Charpentier, B., Bandeiras, T. M., Manival, X., Hernandez-Alba, O., Cianferani, S.. 2023-05-31. Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level. https://doi.org/10.1101/2023.05.30.542861

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