Hydrophilic Interaction Liquid Chromatography at Subzero-Temperature for Hydrogen-Deuterium Exchange Mass Spectrometry

Published

November 6, 2023

Author(s)

Kyle Anderson, Jeffrey W. Hudgens

Abstract

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.

Citation

Journal of the American Society for Mass Spectrometry

Pub Type

Journals

Download Paper

https://doi.org/10.1021/jasms.3c00243

Local Download

Citation

Anderson, K. and Hudgens, J. (2023), Hydrophilic Interaction Liquid Chromatography at Subzero-Temperature for Hydrogen-Deuterium Exchange Mass Spectrometry, Journal of the American Society for Mass Spectrometry, [online], https://doi.org/10.1021/jasms.3c00243, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=956243 (Accessed October 1, 2025)