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

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

Biophys Rep. 2023 Apr 30;9(2):99-111. doi: 10.52601/bpr.2023.230006.

Authors

Qian Meng^{1

2}, Yuan-Li Song^{1

3}, Chen Zhou¹, Han He¹, Naixia Zhang^{1

3}, Hu Zhou^{1

3

4}

Affiliations

¹ Analytical Research Center for Organic and Biological Molecules, State Key Laboratory of Drug Research, State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
² Department of Pharmacology, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
³ University of Chinese Academy of Sciences, Beijing 100049, China.
⁴ School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China.

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.

Keywords: Hsp90N; Hydrogen–deuterium exchange mass spectrometry (HDX-MS); Protein dynamics; Protein–small molecule interactions.