抗原決定位分析不但可更深入瞭解抗原抗體間交互作用，更能促進新型抗體藥物之研發。至今，有許多技術被應用於抗原決定位分析，如X射線晶體學、核磁共振技術、胜肽掃描技術等，各有其優缺點和限制。本研究是首篇以氫氘交換質譜技術進行蛇毒之抗原決定位分析。研究之標的為台灣眼鏡蛇毒液中純化出的神經蛇毒 (Neurotoxin, NTX) 及心臟蛇毒 (Cardiotoxin III, CTX III) 及對該兩者皆具特異性之台灣抗蛇毒馬血清。將抗原及抗原抗體複合物分別進行氫氘交換，在不同的時間點終止反應並使蛋白質變性，最後注入LC-MS系統進行水解和分析。藉由比較目標抗原各胜肽片段在與抗體結合時與未結合時之氫氘交換速率，得以推測可能的抗原決定位位點。由實驗數據可推斷，NTX與CTX III之抗原決定位皆位於β3至β5處，這些區域在抗體結合時較難被溶劑中的氘交換，故具有較低的含氘比例。此外，我們更發現NTX與抗體結合可能導致C54位點有結構上的改變。完成方法開發後，再以越南的抗蛇毒血清驗證該方法的實用性，結果符合預期。根據先前研究顯示，不同的蛇毒抗體得交叉中和蛇毒毒性。基於研發抗體藥物成本高、耗時長，篩選現有抗體對於特定蛇毒的中和效果是另一種選擇。使用自動化的氫氘交換質譜法能迅速有效地做篩選，同時提供蛋白質結構資訊，故本論文所提出之實驗方法對於蛇毒蛋白研究極具發展潛力。

Epitope mapping has been considered a powerful tool that can elucidate binding mechanism and largely facilitates the development of vaccines and drugs. To date, many techniques have been developed for epitope mapping, such as X-ray crystallization, nuclear magnetic resonance, and peptide scanning. Each of them has its own benefits and limitations. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) technique was employed for epitope mapping to probe the binding sites of the short neurotoxin 1 (NTX) and the cardiotoxin III (CTX III) toward particular polyclonal antibodies in Taiwanese bivalent antivenom by comparing the differential rate of deuterium incorporation in their free and Ab-complexed forms. The control samples and the complex samples were incubated in labeling buffer for deuterium labeling respectively. At various time intervals, samples were mixed with quench buffer to quench the H/D exchange. After denaturation, the sample was injected into the LC-MS system for online pepsin digestion and further analysis. The results indicated that the putative binding regions of NTX and CTX III, showing reduced deuterium uptake upon complex formation, were both located within the triple-stranded β sheet. In addition, a conformational change at C54 in NTX was found upon binding. We further investigated the usefulness of this platform by characterizing epitopes of the polyclonal antibodies in Vietnamese snake antivenom. This is the first report that epitopes for antibodies to snake venom toxins were mapped by HDX-MS approach. It can be expected that HDX-MS will be a rapid and efficient method for the evaluation of the cross-neutralization of NTX and CTX III by any antibodies raised by other snake venoms and provides valuable information on conformational changes induced by protein-protein interactions.

1. Timmis, J.; Neal, M.; Hunt, J., An artificial immune system for data analysis. Bio Systems 2000, 55 (1-3), 143-50.
2. Alama, A.; Bruzzo, C.; Cavalieri, Z.; Forlani, A.; Utkin, Y.; Casciano, I.; Romani, M., Inhibition of the nicotinic acetylcholine receptors by cobra venom alpha-neurotoxins: is there a perspective in lung cancer treatment? PloS one 2011, 6 (6), e20695.
3. Guenneugues, M.; Drevet, P.; Pinkasfeld, S.; Gilquin, B.; Menez, A.; Zinn-Justin, S., Picosecond to hour time scale dynamics of a "three finger" toxin: correlation with its toxic and antigenic properties. Biochemistry 1997, 36 (51), 16097-108.
4. Chien, K. Y.; Chiang, C. M.; Hseu, Y. C.; Vyas, A. A.; Rule, G. S.; Wu, W., Two distinct types of cardiotoxin as revealed by the structure and activity relationship of their interaction with zwitterionic phospholipid dispersions. The Journal of biological chemistry 1994, 269 (20), 14473-83.
5. Kumar, T. K.; Lee, C. S.; Yu, C., A case study of cardiotoxin III from the Taiwan cobra (Naja naja atra). Solution structure and other physical properties. Advances in experimental medicine and biology 1996, 391, 115-29.
6. Tsetlin, V., Snake venom alpha-neurotoxins and other 'three-finger' proteins. European journal of biochemistry 1999, 264 (2), 281-6.
7. Kessler, P.; Marchot, P.; Silva, M.; Servent, D., The three-finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions. Journal of Neurochemistry 2017, 142, 7-18.
8. Kini, R. M.; Doley, R., Structure, function and evolution of three-finger toxins: mini proteins with multiple targets. Toxicon : official journal of the International Society on Toxinology 2010, 56 (6), 855-67.
9. Nirthanan, S.; Gopalakrishnakone, P.; Gwee, M. C.; Khoo, H. E.; Kini, R. M., Non-conventional toxins from Elapid venoms. Toxicon : official journal of the International Society on Toxinology 2003, 41 (4), 397-407.
10. Nirthanan, S.; Charpantier, E.; Gopalakrishnakone, P.; Gwee, M. C.; Khoo, H. E.; Cheah, L. S.; Bertrand, D.; Kini, R. M., Candoxin, a novel toxin from Bungarus candidus, is a reversible antagonist of muscle (alphabetagammadelta ) but a poorly reversible antagonist of neuronal alpha 7 nicotinic acetylcholine receptors. The Journal of biological chemistry 2002, 277 (20), 17811-20.
11. Ojeda, P. G.; Ramirez, D.; Alzate-Morales, J.; Caballero, J.; Kaas, Q.; Gonzalez, W., Computational Studies of Snake Venom Toxins. Toxins 2017, 10 (1).
12. Yang, C. C.; Chang, C. C.; Hayashi, K.; Suzuki, T., Amino acid composition and end group analysis of cobrotoxin. Toxicon : official journal of the International Society on Toxinology 1969, 7 (1), 43-47.
13. Yang, C. C.; Yang, H. J.; Chiu, R. H. C., The position of disulfide bonds in cobrotoxin. Biochimica et Biophysica Acta (BBA) - Protein Structure 1970, 214 (2), 355-363.
14. Sivaraman, T.; Kumar, T. K.; Yu, C., Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics. Biochemistry 1999, 38 (31), 9899-905.
15. Yu, C.; Bhaskaran, R.; Chuang, L. C.; Yang, C. C., Solution conformation of cobrotoxin: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study. Biochemistry 1993, 32 (9), 2131-6.
16. Bhaskaran, R.; Huang, C. C.; Chang, D. K.; Yu, C., Cardiotoxin III from the Taiwan cobra (Naja naja atra). Determination of structure in solution and comparison with short neurotoxins. Journal of molecular biology 1994, 235 (4), 1291-301.
17. Gabrielli, E.; Pericolini, E.; Cenci, E.; Ortelli, F.; Magliani, W.; Ciociola, T.; Bistoni, F.; Conti, S.; Vecchiarelli, A.; Polonelli, L., Antibody complementarity-determining regions (CDRs): a bridge between adaptive and innate immunity. PloS one 2009, 4 (12), e8187.
18. Yan, Y.; Wei, H.; Fu, Y.; Jusuf, S.; Zeng, M.; Ludwig, R.; Krystek, S. R., Jr.; Chen, G.; Tao, L.; Das, T. K., Isomerization and Oxidation in the Complementarity-Determining Regions of a Monoclonal Antibody: A Study of the Modification-Structure-Function Correlations by Hydrogen-Deuterium Exchange Mass Spectrometry. Analytical chemistry 2016, 88 (4), 2041-50.
19. Reid, R.; Roberts, F.; MacDuff, E., Pathology Illustrated E-Book. Elsevier Health Sciences: 2011.
20. Verma, A.; Singh, A., Animal biotechnology: models in discovery and translation. Academic Press: 2013.
21. Köhler, G.; Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256 (5517), 495.
22. Lipman, N. S.; Jackson, L. R.; Trudel, L. J.; Weis-Garcia, F., Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR journal 2005, 46 (3), 258-68.
23. Barh, D.; Azevedo, V., Omics Technologies and Bio-engineering: Volume 1: Towards Improving Quality of Life. Academic Press: 2017.
24. Stills, H. F., Chapter 11 - Polyclonal Antibody Production. In The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents, Academic Press: Boston, 2012; pp 259-274.
25. Hanly, W. C.; Artwohl, J. E.; Bennett, B. T., Review of polyclonal antibody production procedures in mammals and poultry. ILAR journal 1995, 37 (3), 93-118.
26. Gutiérrez, J. M.; Rojas, E.; Quesada, L.; León, G.; Núñez, J.; Laing, G. D.; Sasa, M.; Renjifo, J.; Nasidi, A.; Warrell, D. A., Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 2005, 99 (6), 468-475.
27. Hsu, Y.-L.; Wu, C.-J.; Chou, T.-C.; Hsieh, W.-C.; Cheng, Y.-F.; Chiang, J.-R., Retrospection and prospection for manufacturing of snake antivenins in Taiwan. Epidemiology Bulletin 2013, 29 (6), 50-56.
28. Chippaux, J.-P.; Goyffon, M., Production and use of snake antivenin. TU AT, Handbook of natural toxins 1991, 5.
29. Andrew, S. M.; Titus, J. A., Fragmentation of Immunoglobulin G. In Current Protocols in Immunology, John Wiley & Sons, Inc.: 2001.
30. Chippaux, J. P., [Guidelines for the production, control and regulation of snake antivenom immunoglobulins]. Biologie aujourd'hui 2010, 204 (1), 87-91.
31. Barlow, D. J.; Edwards, M. S.; Thornton, J. M., Continuous and discontinuous protein antigenic determinants. Nature 1986, 322 (6081), 747-8.
32. Specter, S., Chemistry of Antigen-Antibody Interactions. In xPharm: The Comprehensive Pharmacology Reference, Elsevier: New York, 2007; pp 1-3.
33. Yokota, A.; Tsumoto, K.; Shiroishi, M.; Kondo, H.; Kumagai, I., The Role of Hydrogen Bonding via Interfacial Water Molecules in Antigen-Antibody Complexation THE HyHEL-10-HEL INTERACTION. Journal of Biological Chemistry 2003, 278 (7), 5410-5418.
34. Ganea, D., ImmunoBiology: The Immune System in Health and Diseases. BioScience 1996, 46 (3), 215-216.
35. Reverberi, R.; Reverberi, L., Factors affecting the antigen-antibody reaction. Blood transfusion 2007, 5 (4), 227.
36. Fukunaga, A.; Tsumoto, K., Improving the affinity of an antibody for its antigen via long-range electrostatic interactions. Protein Engineering, Design & Selection 2013, 26 (12), 773-780.
37. Gershoni, J. M.; Roitburd-Berman, A.; Siman-Tov, D. D.; Tarnovitski Freund, N.; Weiss, Y., Epitope mapping: the first step in developing epitope-based vaccines. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy 2007, 21 (3), 145-56.
38. Ahmad, T. A.; Eweida, A. E.; Sheweita, S. A., B-cell epitope mapping for the design of vaccines and effective diagnostics. Trials in Vaccinology 2016, 5, 71-83.
39. Potocnakova, L.; Bhide, M.; Pulzova, L. B., An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction. Journal of Immunology Research 2016, 2016, 6760830.
40. Domina, M.; Lanza Cariccio, V.; Benfatto, S.; Venza, M.; Venza, I.; Donnarumma, D.; Bartolini, E.; Borgogni, E.; Bruttini, M.; Santini, L.; Midiri, A.; Galbo, R.; Romeo, L.; Patane, F.; Biondo, C.; Norais, N.; Masignani, V.; Teti, G.; Felici, F.; Beninati, C., Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries. PloS one 2016, 11 (8), e0160702.
41. Castro, K. L.; Duarte, C. G.; Ramos, H. R.; Machado de Avila, R. A.; Schneider, F. S.; Oliveira, D.; Freitas, C. F.; Kalapothakis, E.; Ho, P. L.; Chavez-Olortegui, C., Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom. Toxicon : official journal of the International Society on Toxinology 2015, 93, 51-60.
42. Abbott, W. M.; Damschroder, M. M.; Lowe, D. C., Current approaches to fine mapping of antigen–antibody interactions. Immunology 2014, 142 (4), 526-535.
43. Ultsch, M.; Bevers, J.; Nakamura, G.; Vandlen, R.; Kelley, R. F.; Wu, L. C.; Eigenbrot, C., Structural Basis of Signaling Blockade by Anti-IL-13 Antibody Lebrikizumab. Journal of molecular biology 2013, 425 (8), 1330-1339.
44. Lill, J. R.; Sandoval, W., Analytical Characterization of Biotherapeutics. John Wiley & Sons: 2017.
45. Lu, X.; DeFelippis, M. R.; Huang, L., Linear epitope mapping by native mass spectrometry. Analytical Biochemistry 2009, 395 (1), 100-107.
46. Merk, A.; Bartesaghi, A.; Banerjee, S.; Falconieri, V.; Rao, P.; Davis, M.; Pragani, R.; Boxer, M.; Earl, L. A.; Milne, J. L. S.; Subramaniam, S., Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery. Cell 2016, 165 (7), 1698-1707.
47. Casina, V. C.; Hu, W.; Mao, J. H.; Lu, R. N.; Hanby, H. A.; Pickens, B.; Kan, Z. Y.; Lim, W. K.; Mayne, L.; Ostertag, E. M.; Kacir, S.; Siegel, D. L.; Englander, S. W.; Zheng, X. L., High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome. Proceedings of the National Academy of Sciences of the United States of America 2015, 112 (31), 9620-5.
48. Demmer, J. K.; Rupprecht, F. A.; Eisinger, M. L.; Ermler, U.; Langer, J. D., Ligand binding and conformational dynamics in a flavin‐based electron‐bifurcating enzyme complex revealed by Hydrogen–Deuterium Exchange Mass Spectrometry. FEBS letters 2016, 590 (24), 4472-4479.
49. Prądzińska, M.; Behrendt, I.; Astorga-Wells, J.; Manoilov, A.; Zubarev, R. A.; Kołodziejczyk, A. S.; Rodziewicz-Motowidło, S.; Czaplewska, P., Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C. Amino acids 2016, 48 (12), 2809-2820.
50. Deng, B.; Lento, C.; Wilson, D. J., Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development – A review. Analytica Chimica Acta 2016, 940, 8-20.
51. Gallagher, E. S.; Hudgens, J. W., Mapping protein–ligand interactions with proteolytic fragmentation, hydrogen/deuterium exchange-mass spectrometry. In Methods in enzymology, Elsevier: 2016; Vol. 566, pp 357-404.
52. Jansson, E. T.; Lai, Y. H.; Santiago, J. G.; Zare, R. N., Rapid Hydrogen-Deuterium Exchange in Liquid Droplets. Journal of the American Chemical Society 2017, 139 (20), 6851-6854.
53. Zhou, B.; Zhang, Z. Y., Application of hydrogen/deuterium exchange mass spectrometry to study protein tyrosine phosphatase dynamics, ligand binding, and substrate specificity. Methods (San Diego, Calif.) 2007, 42 (3), 227-33.
54. Chen, G., Characterization of protein therapeutics using mass spectrometry. Springer: 2014.
55. Gallagher, E. S.; Hudgens, J. W., Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry. Methods in enzymology 2016, 566, 357-404.
56. Walters, B. T.; Ricciuti, A.; Mayne, L.; Englander, S. W., MINIMIZING BACK EXCHANGE IN THE HYDROGEN EXCHANGE - MASS SPECTROMETRY EXPERIMENT. Journal of the American Society for Mass Spectrometry 2012, 23 (12), 2132-2139.
57. Skinner, J. J.; Lim, W. K.; Bédard, S.; Black, B. E.; Englander, S. W., Protein hydrogen exchange: Testing current models. Protein Science 2012, 21 (7), 987-995.
58. Sivaraman, T.; Kumar, T. K. S.; Yu, C., Investigation of the Structural Stability of Cardiotoxin Analogue III from the Taiwan Cobra by Hydrogen− Deuterium Exchange Kinetics. Biochemistry 1999, 38 (31), 9899-9905.
59. Mathes, T.; Kennis, J., Optogenetic Tools in the Molecular Spotlight. Frontiers in molecular biosciences 2016, 3, 14.
60. Boone, C.; Adamec, J., 10 - Top-Down Proteomics A2 - Ciborowski, P. In Proteomic Profiling and Analytical Chemistry (Second Edition), Silberring, J., Ed. Elsevier: Boston, 2016; pp 175-191.
61. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray ionization for mass spectrometry of large biomolecules. Science (New York, N.Y.) 1989, 246 (4926), 64-71.
62. Wang, L.; Ryan, A. J., 1 - Introduction to electrospinning. In Electrospinning for Tissue Regeneration, Woodhead Publishing: 2011; pp 3-33.
63. Wilm, M., Principles of Electrospray Ionization. Molecular & Cellular Proteomics : MCP 2011, 10 (7), M111.009407.
64. Dass, C., 9.12 - Mass Spectrometry: Structure Determination of Proteins and Peptides. In Comprehensive Natural Products II, Elsevier: Oxford, 2010; pp 457-496.
65. Liu, B. S.; Wu, W. G.; Lin, M. H.; Li, C. H.; Jiang, B. R.; Wu, S. C.; Leng, C. H.; Sung, W. C., Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom. Toxins 2017, 10 (1).