title: Determination of protease subsite preference on SPOT peptide array by fluorescence quenching-based assay.
abstract: A peptide SPOT array was synthesized on a glass chip and used to determine protease subsite preference. To synthesize a peptide array for positional scanning, the ratio of the isokinetic concentration was determined for every Fmoc-amino acid except Cys. Based on this ratio, a peptide array consisting of Dabcyl-X-X-P(2)-Arg-X-X-X-Lys(FITC) (X: equimolar mixture of 19 amino acids, P(2): one of 19 amino acids) was synthesized on a chitosan-grafted glass chip. Subsequently, the peptide substrates on the array were hydrolyzed by thrombin to screen for subsite specificity using a fluorescence quenching-based assay. The P(2) subsite specificity of thrombin was screened by the fluorescence images obtained after hydrolysis. Pro at the P(2) subsite showed the highest specificity for thrombin based on both the fluorescence quenching-based assay and the solution phase assay. From these results, we confirmed that our mixture-based peptide SPOT array format on the chitosan-grafted glass chips could be used to determine protease subsite preference.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Seoul 151-747, Korea.
authors.forename: Do-Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Dong-Sik
authors.initials: DS
authors.lastname: Shin
authors.forename: Yoon-Sik
authors.initials: YS
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005453:Fluorescence; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D040081:Protein Array Analysis
chemical_list: D010455:Peptides; D010447:Peptide Hydrolases