Epitope Mapping of Antibodies against Neurotoxin (NTX) and Cardiotoxin III (CTX III) by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

• Main Authors: Chen, Wei-Ya, 陳薇雅
• Other Authors: Chen, Sung-Fang
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/pgsdgt

碩士 === 國立臺灣師範大學 === 化學系 === 106 === 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.