Development 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 that can be cooled to subzero temperatures. <i>HDXchip</i> comprises a sample loading module with an immobilized enzyme microreactor (IMER) for on-chip pepsin proteolysis and a module packed with reversed-phase material for on-chip desalting and reversed-phase chromatography. The chip thus integrates the hallmarks of the classical HDX-MS workflow in a low-cost, low-volume microfluidic format. Furthermore, the small size of the chip allows for efficient localized cooling of parts of the chip to subzero temperatures by a Peltier module. We show that <i>HDXchip</i> allows for HDX-MS analysis of model peptides and intact proteins at significantly lowered back-exchange compared to a conventional commercially available UPLC-based HDX-MS setup. Furthermore, we show that the chip is capable of local HDX-MS analysis of hemoglobin with good sensitivity, sequence coverage, repeatability, and low back-exchange. Our results demonstrate the potential of integrating the HDX-MS workflow, including proteolysis and separation, with subzero temperature cooling, on a microchip.