Journal of Proteomics & Bioinformatics

Journal of Proteomics & Bioinformatics
Open Access

ISSN: 0974-276X

+44 1223 790975

Abstract

A Novel Mass Spectrometric Epitope Mapping Approach without Immobilization of the Antibody

R. 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 for the determination of an epitope on RA33 against which a monoclonal antibody was directed identified the epitope sequence (85IDGRVVEPKRA95) using MS/MS peptide sequencing. Advantages of this approach include low sample consumption, few handling steps, and short duration of analysis. With our method we are ultimately aiming at developing a screening procedure to identify major epitopes in patients that may be suitable in the future for stratification of patients, needed for personalized therapies.

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