Analysis of the Antigen Binding Site of Anti-Deoxycholate Monoclonal Antibody Using a Novel Affinity Labeling Reagent, Acyl Adenylate

Nariyasu Mano, Yoko Nagaya, Shuntaro Saito, Norihiro Kobayashi, Junichi Goto

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site.

Original languageEnglish
Pages (from-to)2041-2048
Number of pages8
JournalBiochemistry
Volume43
Issue number7
DOIs
Publication statusPublished - 2004 Feb 24

ASJC Scopus subject areas

  • Biochemistry

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