1. [Identification of the epitope of von Willebrand factor recognized by monoclonal antibody SZ-125 with immune-affinity mass spectrometry].
- Author
-
Li X, Wang F, Shen F, Zhao Y, and Jiang M
- Subjects
- Amino Acid Sequence, Molecular Sequence Data, Mutagenesis, Proteolysis, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins immunology, Recombinant Proteins metabolism, Time Factors, Trypsin metabolism, von Willebrand Factor chemistry, von Willebrand Factor genetics, von Willebrand Factor metabolism, Antibodies, Monoclonal immunology, Chromatography, Affinity methods, Epitope Mapping methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, von Willebrand Factor immunology
- Abstract
Objective: To identify the epitope of von Willebrand factor (vWF) recognized by monoclonal antibody SZ-125 (mAb SZ-125) using immune-affinity matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay in combination with peptide synthesis and amino acid site-mutagenesis technology., Methods: Recombinant vWF A3 domain (rVWF A3) was directly affinity bound to SZ-125 antibody beads and proselytized by trypsin. The digested peptide fragments were then measured using MALDI-TOF-MS. The detected peptide sequence by MALDI-TOF-MS was synthesized and several amino-acids in it were mutated to test its affinity with mAb SZ-125., Results: The epitope of rVWF A3 recognized by SZ-125 was identified to be the peptide fragment(1001);EGGPSQIGDALGFAVR(1016);. Synthesized peptide NH2;-EGGPSQIGDALGFAVR-COOH could bind to mAb SZ125., Results: of site-directed mutagenesis revealed that amino acids E1001, F1013, V1015 and R1016 played critical roles in the binding between mAb SZ-125 and rVWF A3., Conclusion: The epitope of rVWF A3 recognized by mAb SZ-125 has been accurately confirmed using immune-affinity mass spectrometry in combination with peptide synthesis and site-directed mutagenesis of special amino acids.
- Published
- 2013