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Updated: Jul 11, 2025

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry
Published on: June 8, 2021
Hydrophilic Interaction Liquid Chromatography at Subzero Temperature for Hydrogen-Deuterium Exchange Mass
Kyle W Anderson1,2, Jeffrey W Hudgens1,2
1Bioprocess Measurements Group, Biomolecular Measurement Division, National Institute of Standards and Technology, Rockville, Maryland 20850, United States.
None:
Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen-deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) at -30 °C, but high backpressures limited flow rates and required materials and equipment rated for very high pressures. Here, we report the design and performance of a dual-enzyme HDX-MS analysis instrument comprising a RPLC trap column and a hydrophilic interaction liquid chromatography (HILIC) analytical column in a two-dimensional RPLC-HILIC configuration at subzero temperature. During operation at -30 °C, the HILIC column manifests greatly reduced backpressure, which enables faster analytical flow rates and the use of materials rated for lower maximum pressures. The average peptide eluted from a HILIC column during a 40 min gradient at -30 °C contained ≈13% more deuterium than peptides eluted from a tandem RPLC-RPLC apparatus using a conventional 8 min gradient at 0 °C. A subset of peptides eluted from the HILIC apparatus contained ≈24% more deuterium.
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