title: Immunoaffinity extraction of a peptide modified by a small molecule.
abstract: We investigated the affinity extraction conditions required to isolate peptide fragments modified with small molecules using an antibody that has a high affinity for the target small molecule. Investigation of antibody conformation and the retention behavior of the modified peptides on an immunosorbent matrix demonstrated the importance in efficient extraction of both the dissociation of hydrophobic interactions and the breakdown of the antibody conformation. Hydrophobic interactions, which anchor the small ligand to the paratope, were retained even when the three-dimensional structure of the antibody disintegrated in an acidic solution. For efficient extraction of a target peptide modified by a small molecule, it is therefore important to use an acidic solvent containing an organic modifier such as methanol at a concentration greater than 40% (v/v). We demonstrated the feasibility of this immunoaffinity extraction by application of this procedure to the analysis of modified peptide fragments obtained from a digestion of human serum albumin. The peptide fragments were affinity labeled with chenodeoxycholyl adenylate for analysis of the chenodeoxycholate binding site. This purification method could isolate the low levels of modified peptide contained in the reaction mixture, despite the presence of appreciable quantities of unlabeled peptide fragments.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
authors.forename: Nariyasu
authors.initials: N
authors.lastname: Mano
authors.forename: Kohei
authors.initials: K
authors.lastname: Abe
authors.forename: Junichi
authors.initials: J
authors.lastname: Goto
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000906:Antibodies; D010455:Peptides