High-Precision, Gas-Phase Hydrogen/Deuterium-Exchange Kinetics by Mass Spectrometry Enabled by Exchange Standards

摘要

Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium exchange (gHDX), MS is also capable of probing the structure of ions. However, gHDX kinetics can depend strongly on many factors, including laboratory temperature, instrumental conditions, and instrument platform selection. These effects can lead to high variability with gHDX measurements, which has hindered the broader adoption of gHDX for structural MS. Here we introduce an approach for standardizing gHDX measurements using cosampled standards. Quantifying the exchange kinetics for analytes relative to the exchange kinetics of the standards results in greater accuracy and precision than the underlying absolute measurements. The standardization was found to be effective for several types of analytes including small molecules and intact proteins. A subset of analytes showed deviations in their standardized exchange profiles that are attributed to field heating and the concomitant conformational isomerization. Inclusion of helium during the gHDX process for collisional cooling helps mitigate such variations in exchange kinetics related to ion heating. We anticipate that the outcomes of this research will enable the broader use of gHDX in MS-based workflows for molecular identification and isomer differentiation.