title: Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.
abstract: To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, β-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 μM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.
authors.affiliation: Laboratory of Proteomic Sciences, 21st Century COE Program, Kyoto Pharmaceutical University, Kyoto, Japan; Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan; Laboratory of Next Generation Drug Development, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan; Protein Research Group, RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.forename: Mamiko
authors.initials: M
authors.lastname: Nakato
authors.forename: Takaaki
authors.initials: T
authors.lastname: Mizuguchi
authors.forename: Shinji
authors.initials: S
authors.lastname: Wada
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Uchimura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kataoka
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hirota
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
mesh_terms: D002147:Calmodulin; D019075:Electrophoresis, Capillary / Q000379:methods*; D008956:Models, Chemical; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine
keywords: ACE; Mass spectrometric detection; Plug-plug technique; Screening of calmodulin-binding peptides; Selection from mixed peptides