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

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.

Significance of Histidine Hydrogen–Deuterium Exchange Mass Spectrometry in Protein Structural Biology

“…The major technical challenge of amide-HDX-MS is the fast back exchange from deuterium to proton, which can occur during protein digestion and mass spectrometry analysis. This reaction is fast; the half-life for the back-exchange reaction is about 1 h at pH 2.5 and at 0 • C [46,47], necessitating completion of the digestion of proteins at acidic pH and analysis of the resulting peptides with LC-MS/MS in approximately 1 h. In contrast, the rate of His-HDX is much slower (t 1/2 = 1.9 days at 37 • C) [34], allowing for relatively long protein digestion at physiological pH at 37 • C using proteases, which work at physiological pH. Additionally, in amide-HDX-MS, the LC-MS separation of peptides is conducted at low temperatures (close to 0 • C) to avoid back exchange.…”