A hydrogen–deuterium exchange mass spectrometry-based protocol for protein–small molecule interaction analysis

Qian Meng; Yuan-Li Song; Chen Zhou; Han He; Naixia Zhang; Hu Zhou

doi:10.52601/bpr.2023.230006

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Biophysics Reports ›› 2023, Vol. 9 ›› Issue (2) : 99-111. DOI: 10.52601/bpr.2023.230006

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A hydrogen–deuterium exchange mass spectrometry-based protocol for protein–small molecule interaction analysis

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Abstract

Protein–small molecule interaction is vital in regulating protein functions and controlling various cellular processes. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein–small molecule interactions, however, to accurately probe the conformational dynamics of the protein upon small molecule binding, the HDX-MS experimental conditions should be carefully controlled and optimized. Here, we present the detailed continuous-labeling, bottom-up HDX-MS protocol for studying protein–small molecule interactions. We took a side-by-side HDX kinetics comparison of the Hsp90N protein with or without the treatment of small molecules (i.e., Radicicol, Geldanamycin) for displaying conformational changes induced by molecular interactions between Hsp90N and small molecules. Our sensitive and robust experimental protocol can facilitate the novice to quickly carry out the structural characterization of protein–small molecule interactions.

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Hydrogen–deuterium exchange mass spectrometry (HDX-MS) / Protein dynamics / Protein–small molecule interactions / Hsp90N

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Qian Meng, Yuan-Li Song, Chen Zhou, Han He, Naixia Zhang, Hu Zhou. A hydrogen–deuterium exchange mass spectrometry-based protocol for protein–small molecule interaction analysis. Biophysics Reports, 2023, 9(2): 99‒111 https://doi.org/10.52601/bpr.2023.230006

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Acknowledgements

This study is supported by the National Key Research and Development Program (2022YFA1302900), Shanghai Young Excellent Academic Leader Program (20XD1424900), Shanghai Municipal Science and Technology Major Project, the National Natural Science Foundation of China (32171220, 22107111, 21977105), and the Youth Innovation Promotion Association CAS (2022284). We would like to thank the Institutional Center for Shared Technologies and Facilities of Shanghai Institute of Materia Medica for all the technical support.