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Chromatography at -30 °C for Reduced Back-Exchange, Reduced Carryover, and Improved Dynamic Range for Hydrogen-Deuterium Exchange Mass Spectrometry



Kyle Anderson, Jeffrey W. Hudgens


For hydrogen-deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during protein analyses should be minimized to promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 and 0 °C can lose > 30 % of the deuterium marker within 15 minutes of sample injection. This report describes the architecture and performance of a dual-enzyme, HDX-MS instrument that conducts liquid chromatography (LC) separations at subzero temperature, thereby reducing back-exchange and supporting longer LC separations with improved chromatographic resolution. LC separations of perdeuterated, fully reduced, iodoacetamide-treated BSA protein digest standard peptides were performed at (0, 10, 20, 30) °C in ethylene glycol (EG)/H2O mixtures. The average peptide eluted during a 40 min gradient at -30 °C contained (17 ± 7) % more deuterium than peptides eluted with a conventional 8 min gradient at 0 °C. A subset of peptides exhibited (26 ± 2) % improvement. Although chromatographic peaks shift with EG concentration and temperature, the apparatus elutes unbroadened LC peaks. Electrospray ion intensity does not decline with increasing EG fraction. To minimize bias from sample carryover, the fluidic circuits allow flush and backflush cleaning of all enzyme and LC columns. The system can perform LC separations and clean enzyme columns simultaneously. Temperature zones are controlled ±0.058 °C. The potential of increased sensitivity by mixing acetonitrile with the analytical column effluent was also examined.
Journal of the American Society for Mass Spectrometry


biochemistry, chromatography, hydrogen-deuterium exchange, low temperature, mass spectrometry, peptide analysis, protein, structure-function biology


Anderson, K. and Hudgens, J. (2022), Chromatography at -30 °C for Reduced Back-Exchange, Reduced Carryover, and Improved Dynamic Range for Hydrogen-Deuterium Exchange Mass Spectrometry, Journal of the American Society for Mass Spectrometry, [online],, (Accessed April 21, 2024)
Created June 22, 2022, Updated November 29, 2022