Journal of Proteomics & Bioinformatics
Open Access
ISSN: 0974-276X
ISSN: 0974-276X
H.-J. Thiesen
Germany
Research Article
A Novel Mass Spectrometric Epitope Mapping Approach without Immobilization of the Antibody
Author(s): R. F. El-Kased, C. Koy, P. Lorenz, H. Montgomery, K. Tanaka, H.-J. Thiesen and M. O. GlockerR. F. El-Kased, C. Koy, P. Lorenz, H. Montgomery, K. Tanaka, H.-J. Thiesen and M. O. Glocker
The development and application of a mass spectrometry-based epitope mapping procedure in solution and without immobilization of the antibody is described. Antigens were digested with proteases. Then size exclusion micro-column chromatography (SEC) was carried out prior to and upon exposure of the peptide mixtures to monoclonal antibodies. The epitope-containing peptide, affinity bound to the antibody and, thus, forming a stable complex eluted early as did all high-mass components, whereas all unbound low-mass peptides eluted late. Comparison of elution profiles in the presence and absence of the antibody showed a shift only for epitope-bearing peptides, enabling direct identification of the epitope. His-tag containing recombinant antigens Fibrillarin and RA33 were used in combination with an anti-His-tag monoclonal antibody in order to develop the method. Application of this method f.. View More»
DOI:
10.4172/jpb.1000160