biostudies-literatureHansen ABDanmarks Frie ForskningsfondEuropean Research Council12503-12512https://www.ebi.ac.uk/biostudies/studies/S-EPMC12199231biostudies-literatureUnknown97(24)Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for analyzing the conformational dynamics of proteins. Using liquid chromatography coupled to mass spectrometry (LC-MS), the method achieves high sensitivity and the option to measure the HDX either of the intact protein (global analysis) or of small peptide segments of the protein (local analysis) after online pepsin proteolysis. However, HDX-MS is currently limited by the significant cost of the specialized refrigerated UPLC-based chromatographic equipment needed and by the loss of deuterium label from the analyzed protein (30-50%), which still occurs despite cooling parts of the LC-MS system to 0 °C. Here we describe the development of a microfluidic chip (<i>HDXchip</i>) for global or local HDX-MS analysis tAnalytical chemistryDevelopment of a Thiol-ene Microfluidic Chip for Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS).PMC12199231101003052060202740B0602-02100BHamidi MKutter JPHarmash DRand KDHansen ABSticker DSvejdal RRAerts JTStahlhut VBPfalseDevelopment of a Thiol-ene Microfluidic Chip for Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS).Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for analyzing the conformational dynamics of proteins. Using liquid chromatography coupled to mass spectrometry (LC-MS), the method achieves high sensitivity and the option to measure the HDX either of the intact protein (global analysis) or of small peptide segments of the protein (local analysis) after online pepsin proteolysis. However, HDX-MS is currently limited by the significant cost of the specialized refrigerated UPLC-based chromatographic equipment needed and by the loss of deuterium label from the analyzed protein (30-50%), which still occurs despite cooling parts of the LC-MS system to 0 °C. Here we describe the development of a microfluidic chip (<i>HDXchip</i>) for global or local HDX-MS analysis t2025-01-01T00:00:00Z2025 Jun2026-06-02T05:57:35.629Z2026-04-15T03:09:56.567ZS-EPMC121992314048867210.1021/acs.analchem.4c06230