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.
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 beta-amyloid-peptide Abeta (1-40) 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-beta 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.
Bílková
Z
Z
Laboratory of Physical Chemistry, Institute Curie (UMR CNRS/IC 168), Paris Cedex 05, France.
Stefanescu
R
R
Cecal
R
R
Korecká
L
L
Ouzká
S
S
Jezová
J
J
Viovy
J-L
JL
Przybylski
M
M
D017357:Cyclotrons; D004800:Enzymes, Immobilized; D000939:Epitopes / Q000302:isolation & purification; D005583:Fourier Analysis; D007477:Ions; D008280:Magnetics; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification; D014357:Trypsin; D014612:Vaccines / Q000737:chemistry*
D004800:Enzymes, Immobilized; D000939:Epitopes; D007477:Ions; D010455:Peptides; D014612:Vaccines; D014357:Trypsin