Analytical Aspects of Hydrogen Exchange Mass Spectrometry

Abstract

The analytical aspects of measuring hydrogen exchange by mass spectrometry are reviewed. The nature of analytical selectivity in hydrogen exchange is described followed by review of the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in hydrogen exchange mass spectrometry depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that could be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics.

Replication in Bioanalytical Studies with Hdx MS: Aim as High as Possible

“…We have defined [5] five categories or levels of replication in HDX MS experiments: biological, manipulation, labeling, analysis and processing. There is a hierarchy to these replicate levels with biological replication being the highest order and processing replication being lowest order.…”

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

“…(9)(10)(11)(12)(13) The method is , that it has great resolution and sensitivity, that it allows for an accessible mass range of up to 10 6 Da, that it allows high speed analysis and that it is a nonensemble technique; each single spectrum contains extractible information about all discrete coexisting states of the protein of interest. By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control.…”

Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

“…Hydrogen deuterium exchange mass spectrometry (HDX-MS) has proven to be a useful tool for investigating protein conformation, dynamics, and interactions [9,10]. However, a traditional continuous dilution labeling HDX-MS experiment has inherently limited utility for the direct analysis of proteins in high concentration solutions because it requires diluting the protein sample into the same buffer containing deuterium (i.e., D 2 O instead of H 2 O).…”