Epitope extraction technique using a proteolytic magnetic reactor combined with Fourier-transform ion cyclotron resonance mass spectrometry as a tool for the screening of potential vaccine lead peptides

Eur. J. Mass Spectrom. 11, 489–495 (2005)
DOI: 10.1255/ejms.782

Z. Bílková,^a,c R. Stefanescu,^b R. Cecal,^b L. Korecká,^c Š. Ouzká,^c J. Ježová,^c J.-L. Viovy^a and M. Przybylski^b
^aLaboratory of Physical Chemistry, Institute Curie (UMR CNRS/IC 168), Paris Cedex 05, France
^bFaculty of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, Box M 732, 78457 Konstanz, Germany
^cDepartment of Biological and Biochemical Sciences, University of Pardubice, Nám. Čs. Legií 565, 532 16 Pardubice, Czech Republic

ABSTRACT:

Epitope extraction technique is based on the specific digestion of a target protein followed by immunoaffinity isolation of a specific recognition peptide. This technique, in combination with mass spectrometry, has been efficiently used for epitope identification. The major goal of this work was to utilize newly developed enzyme and immunoaffinity magnetic reactors for the epitope extraction procedure and confirm the efficiency of this improved system for epitope screening of proteins. Alginic acid-coated magnetite microparticles with immobilized TPCK-trypsin provided high working efficiency with low non-specific adsorption, digestion time in minutes and low frequency of missed cleavages. The sensitivity and specificity of tryptic fragmentation of the β-amyloid- peptide Aβ (1Ő40) as a model polypeptide was confirmed by Fourier-transform ion cyclotron resonance mass spectrometry analysis. The Sepharose reactor or immunoaffinity magnetic reactors, both with anti-amyloid-β monoclonal antibodies, were used for specific isolation and identification of target peptides. In this way, the epitope extraction technique combined with mass spectrometric analysis is shown to be an excellent base for molecular screening of potential vaccine lead proteins.

Keywords: trypsin, magnetic microspheres, Aβ (1Ő40) peptide, epitope extraction, immunosorbent, FT-ICR-MS, vaccine lead structure

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