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Determination of protease subsite preference on SPOT peptide array by fluorescence quenching‐based assay
Author(s) -
Kim DoHyun,
Shin DongSik,
Lee YoonSik
Publication year - 2012
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.2409
Subject(s) - peptide , chemistry , fluorescence , protease , quenching (fluorescence) , amino acid , chromatography , thrombin , hydrolysis , peptide library , enzyme , peptide sequence , biochemistry , biology , physics , platelet , quantum mechanics , gene , immunology
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. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.