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 (HDXchip) for global or local HDX-MS analysis that can be cooled to subzero temperatures. HDXchip 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 HDXchip 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.氢氘交换质谱（Hydrogen/deuterium exchange mass spectrometry, HDX-MS）是一种用于解析蛋白质构象动力学的高效技术。该方法借助液相色谱-质谱联用（liquid chromatography coupled to mass spectrometry, LC-MS）实现了高灵敏度，且可在在线胃蛋白酶水解后，选择对完整蛋白质进行氢氘交换分析（全局分析），或对蛋白质的小肽段进行分析（局部分析）。 然而当前HDX-MS仍存在两大局限：一是所需的专用冷藏式超高效液相色谱（UPLC）色谱设备成本高昂；二是即便将LC-MS系统的部分组件冷却至0 ℃，分析过程中仍会发生分析蛋白质的氘标记流失（流失率达30%~50%）。 本文介绍了一种可冷却至亚低温的微流控芯片（HDXchip），用于全局或局部HDX-MS分析。HDXchip包含两个模块：其一为搭载固定化酶微反应器（immobilized enzyme microreactor, IMER）的样品加载模块，可实现芯片上的胃蛋白酶水解；其二为填充反相材料的模块，用于芯片上的脱盐与反相色谱分离。该芯片以低成本、低体积的微流控形式，整合了经典HDX-MS工作流程的核心特征。 此外，该芯片体积小巧，可通过帕尔贴模块（Peltier module）对芯片的部分区域实现高效的局部亚低温冷却。 研究结果表明，与传统商用的基于UPLC的HDX-MS装置相比，HDXchip可在显著降低反向交换（back-exchange）的前提下，对模型肽段与完整蛋白质进行HDX-MS分析。 进一步研究显示，该芯片可对血红蛋白（hemoglobin）进行局部HDX-MS分析，且具备良好的灵敏度、序列覆盖度、重复性与低反向交换率。 本研究结果证实，将包括蛋白水解与分离在内的HDX-MS工作流程与亚低温冷却技术集成于微芯片上具有可观的应用潜力。