Hydrogen exchange mass spectrometry for the analysis of protein dynamics

Abstract

Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.

Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors

“…As these experiments were performed as comparisons under identical experimental conditions, no corrections have been made for back-exchange thus the absolute value of each deuterium level is 18-25% higher than plotted based on totally deuterated standards (described in detail in ref. 24). The cumulative error of measuring deuterium uptake in these assays is B±0.20 Da.…”

“…With HX-MS, the relative dynamics within a protein are monitored by measuring the exchange of backbone amide hydrogens with the bulk solvent 24 . First, experimental methods were developed to provide adequate DOT1L catalytic domain protein coverage (498%) following pepsin digestion (Supplementary Fig.…”

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

“…Hydrogen deuterium exchange (HDX) coupled with MS has been extensively and successfully used as a non-covalent labeling tool to study protein structure and dynamics in solution. [1][2][3] This approach reports on a protein's backbone structure, but back-exchange during the various stages of analysis (e.g. LC, MS/MS) can result in the loss of pertinent data and can complicate data interpretation.…”

Isofagomine Induced Stabilization of Glucocerebrosidase

“…[7] Here we probe the effects of IFG-binding in solution on GCase global stability by differential scanning fluorimetry [18][19][20][21][22] and on local dynamics by amide hydrogen/ deuterium exchange coupled with proteolysis and mass spectrometry (H/D-Ex). [23][24][25][26][27][28] The ability to partially restore intracellular trafficking and lysosomal GCase localization of mutant forms of GCase was then inferred from activity assays performed on N370S/N370S and F213I/L444P patient fibro-blasts. These experiments were employed to gain a better understanding of the PC mechanism associated with IFG binding, particularly the relationship between local dynamics, global stability in solution and their effect on lysosomal concentrations of the enzyme.…”