Development and Applications of HDX-NMR and HDX-MS in Protein Structure and Dynamics Research

doi:10.11938/cjmr20253170

Abstract

Abstract:

Hydrogen-deuterium exchange nuclear magnetic resonance (HDX-NMR) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) are key techniques for studying protein structure and dynamics, and have been widely applied to analyzing protein conformational changes in recent years. HDX-NMR provides dynamic information at single amino acid resolution by detecting nuclear magnetic resonance signals after exchange, and is suitable for studying slow exchange regions and conformational changes over long time scales. HDX-MS integrates the advantages of hydrogen-deuterium exchange and high-resolution mass spectrometry, enabling the determination of protein solution structures under near-physiological conditions, and is applicable to complex systems such as macromolecular complexes and membrane proteins. Compared with traditional techniques like X-ray crystallography and cryo-electron microscopy, HDX-MS is characterized by high sensitivity and low sample demand, while HDX-NMR is superior in site resolution and kinetic analysis. Ongoing technological and methodological optimizations are further broadening the application prospects of both techniques in areas such as protein conformational changes and drug screening. This article reviews their principles, procedures, and analytical methods, compares their similarities and differences, discusses their complementarity and integrated applications, and outlines future directions, aiming to provide references for related research.

Key words: HDX-NMR, HDX-MS, protein, structural dynamics

CLC Number:

O482.53

ZHANG Yuanyuan, WANG Pengcheng, LI Tao, HU Rui, YANG Yunhuang, LIU Maili. Development and Applications of HDX-NMR and HDX-MS in Protein Structure and Dynamics Research[J]. Chinese Journal of Magnetic Resonance, 2025, 42(4): 445-456.

Figures/Tables 5

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Table 1

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比较维度	HDX-NMR	HDX-MS
技术原理	HDX后蛋白质分子酰胺键上的H-N信号强度的衰减	HDX后，酶解为肽段，通过MS检测质荷比变化
分辨率	单个氨基酸残基分辨率，定位精确	肽段级别分辨率，具体位点定位依赖酶解位点
结构信息	可提供构象变化、柔性区、相互作用位点等细节	可识别构象变化区域，但精细结构信息有限
实时动态监测	监测某一时间段的蛋白质分子信号的平均变化	需快速淬灭和冷冻，实时性相对较弱
样品处理	冻干重溶于重水，无需酶解，避免酶解引入偏差	需蛋白酶消化，可能引入序列覆盖度不全或选择性偏差
适用蛋白大小	分子量限制，通常适于 < 30 kDa的中小蛋白	可应用于大分子蛋白甚至多蛋白复合物
样品浓度要求	浓度要求高（通常 > 0.1 mmol/L）	浓度要求低，适合高通量筛选
检测灵敏度	灵敏度相对较低	MS灵敏度高，适合复杂样品和痕量检测
数据解析难度	谱图复杂，解析过程需要专业知识，周期较长	数据分析流程成熟，软件支持广泛，适合标准化处理
非破坏性	样品可重复使用，适合长期跟踪观察	酶解和MS过程不可逆，样品不可回收
典型应用	蛋白-配体结合、构象动态研究、弱相互作用分析	构象筛选、蛋白质稳定性、复合物交互界面快速分析