HDX-NMR与HDX-MS在蛋白质结构动力学研究中的应用与进展
收稿日期: 2025-06-13
网络出版日期: 2025-09-16
基金资助
中国科学院基础与交叉前沿科研B类先导专项(XDB0540000);国家自然科学基金资助项目(22374155);国家自然科学基金资助项目(22327901)
Development and Applications of HDX-NMR and HDX-MS in Protein Structure and Dynamics Research
Received date: 2025-06-13
Online published: 2025-09-16
氢氘交换核磁共振（HDX-NMR）与氢氘交换质谱（HDX-MS）是研究蛋白质结构与动力学的关键技术，近年广泛应用于解析蛋白质构象变化．HDX-NMR通过检测交换后的核磁共振信号，提供单氨基酸分辨率的动态信息，适用于缓慢交换区域及长时间尺度构象变化研究．HDX-MS整合氢氘交换与高分辨率质谱优势，可在近生理条件下测定蛋白质溶液态结构，适用于大分子复合体、膜蛋白等复杂体系．相较于X射线晶体学、冷冻电镜等传统技术，HDX-MS以高灵敏度和低样品需求量见长，HDX-NMR则在位点分辨率与动力学分析上更优．随着技术与方法的持续优化，二者在蛋白质构象变化、药物筛选等领域应用前景日益广阔．本文综述其原理、流程及分析方法，比较异同，探讨互补性与整合应用，展望未来方向，为相关研究提供参考．
张媛媛 , 汪鹏程 , 李滔 , 胡锐 , 杨运煌 , 刘买利 . HDX-NMR与HDX-MS在蛋白质结构动力学研究中的应用与进展[J]. 波谱学杂志, 2025 , 42(4) : 445 -456 . DOI: 10.11938/cjmr20253170
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
| [1] | CHAYEN N E. Turning protein crystallisation from an art into a science[J]. Curr Opin Struc Biol, 2004, 14(5): 577-583. |
| [2] | WüTHRICH K. NMR studies of structure and function of biological macromolecules (Nobel lecture)[J]. Angew Chem Int Ed, 2003, 42(29): 3340-3363. |
| [3] | CHENG Y. Single-particle cryo-EM-How did it get here and where will it go[J]. Science, 2018, 361(6405): 876-880. |
| [4] | HENZLER-WILDMAN K, KERN D. Dynamic personalities of proteins[J]. Nature, 2007, 450(7172): 964-972. |
| [5] | SPRANGERS R, KAY L E. Quantitative dynamics and binding studies of the 20S proteasome by NMR[J]. Nature, 2007, 445(7128): 618-622. |
| [6] | NOGALES E, SCHERES S H. Cryo-EM: A unique tool for the visualization of macromolecular complexity[J]. Mol Cell, 2015, 58(4): 677-689. |
| [7] | NGUYEN T T, MARZOLF D R, SEFFERNICK J T, et al. Protein structure prediction using residue-resolved protection factors from hydrogen-deuterium exchange NMR[J]. Structure, 2022, 30(2): 313-320.e3. |
| [8] | PEACOCK R B, KOMIVES E A. Hydrogen/deuterium exchange and nuclear magnetic resonance spectroscopy reveal dynamic allostery on multiple time scales in the serine protease thrombin[J]. Biochemistry, 2021, 60(46): 3441-3448. |
| [9] | HEBLING C M, MORGAN C R, STAFFORD D W, et al. Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry[J]. Anal Chem, 2010, 82(13): 5415-5419. |
| [10] | ZHANG M M, HUANG R Y, BENO B R, et al. Epitope and paratope mapping of PD-1/nivolumab by mass spectrometry-based hydrogen-deuterium exchange, cross-linking, and molecular docking[J]. Anal Chem, 2020, 92(13): 9086-9094. |
| [11] | LU H R, HUANG Q S. Hydrogen/deuterium exchange mass spectrometry and its application in the study of protein-protein interaction[J]. Biophys Rep, 2013, 29(11):811-822. |
| 陆海荣, 黄青山. 氢氘交换质谱技术及其在蛋白质相互作用研究中的应用[J]. 生物物理学报, 2013, 29(11): 811-822. | |
| [12] | HUANG J, LI H L. Application progress of hydrogen deuterium exchange mass spectrometry in structural study of proteins and protein complexes[J]. J Instrum Anal, 2020, 39(1): 57-67. |
| 黄静, 李惠琳. 氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展[J]. 分析测试学报, 2020, 39(1): 57-67. | |
| [13] | MA L Y, SUN H F, LIU J Y, et al. Application of hydrogen-deuterium exchange mass spectrometry technology in the field of protein drug research[J]. Life Sci Instrum, 2021, 19(3): 20-28. |
| 马凌云, 孙浩峰, 刘健仪, 等. 氢氘交换质谱技术在蛋白质类药物研究领域的应用[J]. 生命科学仪器, 2021, 19(3): 20-28. | |
| [14] | KONERMANN L, PAN J, LIU Y H. Hydrogen exchange mass spectrometry for studying protein structure and dynamics[J]. Chem Soc Rev, 2011, 40(3): 1224-1234. |
| [15] | CHU I T, PIELA G J. Using NMR-detected hydrogen-deuterium exchange to quantify protein stability in cosolutes, under crowded conditions in vitro and in cells[J]. Magn Reson Lett, 2023(3): 319-326. |
| [16] | ENGEN J R. Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS[J]. Anal Chem, 2009, 81(19), 7870-7875. |
| [17] | LIU H, WANG D, ZHANG Q, ZHAO Y, et al. Parallel post-translational modification scanning enhancing hydrogen-deuterium exchange-mass spectrometry coverage of key structural regions[J]. Anal Chem, 2019, 91(11): 6976-6980. |
| [18] | SUN H, MA L, WANG L, et al. Research advances in hydrogen-deuterium exchange mass spectrometry for protein epitope mapping[J]. Anal Bioanal Chem, 2021, 413(9): 2345-2359. |
| [19] | WONG Z W, YANG D. Specific protein-urea interactions[J]. Magn Reson Lett, 2022(2): 131-138. |
| [20] | SCHANDA P, BRUTSCHER B. Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds[J]. J Am Chem Soc, 2005, 127(22): 8014-8015. |
| [21] | FENG Y C, AGRAWAL S, YANG C H, et al. The Structural and dynamic insights into the Ala97Ser amyloidogenic mutation in transthyretin[J]. Chem Asian J, 2025, 20(4): e202401438. |
| [22] | YANAKA S, YAGI-UTSUMI M, KATO K, et al. The B domain of protein A retains residual structures in 6 M guanidinium chloride as revealed by hydrogen/deuterium-exchange NMR spectroscopy[J]. Protein Sci, 2023, 32(3): e4569. |
| [23] | BASAK S, NOBREGA R P, TAVELLA D, et al. Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein[J]. Proc Natl Acad Sci U S A, 2019, 116(14): 6806-6811. |
| [24] | SO P K. Hydrogen-deuterium exchange mass spectrometry for probing changes in conformation and dynamics of proteins[J]. Methods Mol Biol, 2021, 2199: 159-173. |
| [25] | MCALLISTER R G, KONERMANN L. Challenges in the interpretation of protein h/d exchange data: a molecular dynamics simulation perspective[J]. Biochemistry, 2015, 54(16): 2683-2692. |
| [26] | ZHANG Z, ZHANG A, XIAO G. Improved protein hydrogen/deuterium exchange mass spectrometry platform with fully automated data processing[J]. Anal Chem, 2012, 84(11): 4942-4949. |
| [27] | YU J, UZUNER U, LONG B, et al. Artificial intelligence-based HDX (AI-HDX) prediction reveals fundamental characteristics to protein dynamics: Mechanisms on SARS-CoV-2 immune escape[J]. iScience, 2023, 26(4): 106282. |
| [28] | MISTARZ U H, BELLINA B, JENSEN P F, et al. UV photodissociation mass spectrometry accurately localize sites of backbone deuteration in peptides[J]. Anal Chem, 2018, 90(2): 1077-1080. |
| [29] | BRODIE N I, HUGUET R, ZHANG T, et al. Top-down hydrogen-deuterium exchange analysis of protein structures using ultraviolet photodissociation[J]. Anal Chem, 2018, 90(5): 3079-3082. |
| [30] | HAYNES C A, KEPPEL T R, MEKONNEN B, et al. Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein[J]. Rapid Commun Mass Spectrom, 2024, 38(5): e9690. |
| [31] | HANSEN A B, SVEJDAL R R, AERTS J T, et al. Development of a thiol-ene microfluidic chip for hydrogen/deuterium exchange mass spectrometry (HDX-MS)[J]. Anal Chem, 2025, 97(24): 12503-12512. |
| [32] | GRIFFITHS D, ANDERSON M, RICHARDSON K, et al. Cyclic ion mobility for hydrogen/deuterium exchange-mass spectrometry applications[J]. Anal Chem, 2024, 96(15): 5869-5877. |
| [33] | WOOD V E, KELLERMAN M A, GROVES K, et al. Investigation of the solid-state interactions in lyophilized human G-CSF using hydrogen-deuterium exchange mass spectrometry[J]. Mol Pharm, 2024, 21(4): 1965-1976. |
| [34] | LAU A M, JIA R, BRADSHAW R T, et al. Structural predictions of the functions of membrane proteins from HDX-MS[J]. Biochem Soc Trans, 2020, 48(3): 971-979. |
| [35] | ST?NDER S, SCARSELLI M, et al. Epitope mapping of polyclonal antibodies by hydrogen-deuterium exchange mass spectrometry (HDX-MS)[J]. Anal Chem, 2021, 93(34): 11669-11678. |
| [36] | CALVARESI V, WROBEL A G, TOPOROWSKA J, et al. Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein[J]. Nat Commun, 2023, 14(1): 1421. |
| [37] | LOU S C, WETZEL S, ZHANG H, et al. The knotted protein UCH-L1 exhibits partially unfolded forms under native conditions that share common structural features with its kinetic folding intermediates[J]. J Mol Biol, 2016, 428(11): 2507-2520. |
| [38] | MOULICK R, DAS R, UDGAONKAR J B. Partially unfolded forms of the prion protein populated under misfolding-promoting conditions: characterization by hydrogen exchange mass spectrometry and NMR[J]. J Biol Chem, 2015, 290(42): 25227-25240. |
| [39] | UZAWA T, NISHIMURA C, AKIYAMA S, et al. Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR[J]. Proc Natl Acad Sci U S A, 2008, 105(37): 13859-13864. |
| [40] | GELENTER M D, WANG T, LIAO S Y, et al. (2)H-(13)C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates[J]. J Biomol NMR, 2017, 68(4): 257-270. |
| [41] | KUWAJIMA K, YAGI-UTSUMI M, YANAKA S, et al. DMSO-quenched H/D-exchange 2D NMR spectroscopy and its applications in protein science[J]. Molecules, 2022, 27(12): 3748. |
| [42] | NASER D, TARASCA M V, SIEBENEICHLER B, et al. High-resolution NMR H/D exchange of human superoxide dismutase inclusion bodies reveals significant native features despite structural heterogeneity[J]. Angew Chem Int Ed, 2022, 61(24): e202112645. |
| [43] | ZHOU H, HE X, XIONG Y, et al. Structural insights into a highly flexible zinc finger module unravel INSM1 function in transcription regulation[J]. Nat Commun, 2025, 16(1): 2162. |
| [44] | HUANG R Y, IACOB R E, SANKARANARAYANAN S, et al. Probing conformational dynamics of tau protein by hydrogen/deuterium exchange mass spectrometry[J]. J Am Soc Mass Spectrom, 2018, 29(1): 174-182. |
| [45] | SEETALOO N, ZACHAROPOULOU M, STEPHENS A D, et al. Millisecond hydrogen/deuterium-exchange mass spectrometry approach to correlate local structure and aggregation in α-synuclein[J]. Anal Chem, 2022, 94(48): 16711-16719. |
| [46] | SHAW A L, SURESH S, PARSON M A H, et al. Structure of calcineurin bound to PI4KA reveals dual interface in both PI4KA and FAM126A[J]. Structure, 2024, 32(11): 1973-1983.e6. |
| [47] | BAGGEN J, JACQUEMYN M, PERSOONS L, et al. TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry[J]. Cell, 2023, 186(16): 3427-3442.e22. |
| [48] | SHAH M B, JANG H H, et al. Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry[J]. Biophys Chem, 2016, 216: 1-8. |
| [49] | LéGER C, PITARD I, SADI M, et al. Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium[J]. BMC Biol, 2022, 20(1): 176. |
| [50] | GRAUSLUND L R, CALVARESI V, PANSEGRAU W, et al. Epitope and paratope mapping by HDX-MS combined with SPR elucidates the difference in bactericidal activity of two anti-NadA monoclonal antibodies[J]. J Am Soc Mass Spectrom, 2021, 32(7): 1575-1582. |
| [51] | HAMACHI T, YANAI N. Recent developments in materials and applications of triplet dynamic nuclear polarization[J]. Prog Nucl Magn Reson Spectrosc, 2024, 142-143: 55-68. |
| [52] | HOBO F, TANIMOTO Y, ENDO Y, et al. 400 MHz/263 GHz ultra-low temperature MAS-DNP using a closed-cycle helium gas cooling system and a solid-state microwave source[J]. J Magn Reson, 2025, 373: 107842. |
| [53] | BAHTI A, TELFAH A, HERGENR?DER R, et al. NMR spectral editing, water suppression, and dipolar decoupling in low-field NMR spectroscopy using optimal control pulses and multiple-pulse sequence[J]. Anal Chem, 2025, 97(4): 1983-1991. |
| [54] | CRAFT DL, SCHUYLER AD. NUS-tool: A unified program for generating and analyzing sample schedules for nonuniformly sampled NMR experiments[J]. J Magn Reson, 2023, 352: 107458. |
| [55] | RAO KAKITA V M, BOPARDIKAR M, KUMAR SHUKLA V, et al. An efficient combination of BEST and NUS methods in multidimensional NMR spectroscopy for high throughput analysis of proteins[J]. RSC Adv, 2018, 8(32): 17616-17621. |
| [56] | BECKER J, KOOS M R M, SCHULZE-SüNNINGHAUSEN D, et al. ASAP-HSQC-TOCSY for fast spin system identification and extraction of long-range couplings[J]. J Magn Reson, 2019, 300: 76-83. |
/
| 〈 |
|
〉 |