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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
Author(s) -
Saito Kazuki,
Nakato Mamiko,
Mizuguchi Takaaki,
Wada Shinji,
Uchimura Hiromasa,
Kataoka Hiroshi,
Yokoyama Shigeyuki,
Hirota Hiroshi,
Kiso Yoshiaki
Publication year - 2014
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300339
Subject(s) - calmodulin , peptide , chemistry , mastoparan , chromatography , protease , biochemistry , enzyme , g protein , receptor
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 K d 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.