Epitope structure of the carbohydrate recognition domain of asiaglycoprotein receptor to a monoclonal antibody revealed by high resolution proteolytic excision mass spectrometry

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2011
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Stefanescu, Raluca
Born, Rita
Ernst, Beat
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Journal of The American Society for Mass Spectrometry. 2011, 22(1), pp. 148-157. ISSN 1044-0305. eISSN 1879-1123. Available under: doi: 10.1007/s13361-010-0010-y
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Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as entry site into hepatocytes by hepatitis A and B virus, and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LCMS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of 2 intra-molecular disulfide bridges (7 Cys residues), and a Cysmercaptoethanol adduct formed by treatment with ß-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cysalkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide activated Sepharose. Epitope- excision and - extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides, (5-16) and (17-23) which showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

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540 Chemie
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Asialoglycoprotein receptor, H1CRD protein, epitope structure, proteolytic epitope excision, high resolution mass spectrometry
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ISO 690STEFANESCU, Raluca, Rita BORN, Adrian MOISE, Beat ERNST, Michael PRZYBYLSKI, 2011. Epitope structure of the carbohydrate recognition domain of asiaglycoprotein receptor to a monoclonal antibody revealed by high resolution proteolytic excision mass spectrometry. In: Journal of The American Society for Mass Spectrometry. 2011, 22(1), pp. 148-157. ISSN 1044-0305. eISSN 1879-1123. Available under: doi: 10.1007/s13361-010-0010-y
BibTex
@article{Stefanescu2011-01Epito-1012,
  year={2011},
  doi={10.1007/s13361-010-0010-y},
  title={Epitope structure of the carbohydrate recognition domain of asiaglycoprotein receptor to a monoclonal antibody revealed by high resolution proteolytic excision mass spectrometry},
  number={1},
  volume={22},
  issn={1044-0305},
  journal={Journal of The American Society for Mass Spectrometry},
  pages={148--157},
  author={Stefanescu, Raluca and Born, Rita and Moise, Adrian and Ernst, Beat and Przybylski, Michael}
}
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