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.

Dissecting MMP P10′ and P11′ subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library

“…Even a 10-fold error in a mixture equimolarity resulted in less than a 2-fold error in the screening result (IC 50 value) (67), which does not eliminate the usefulness of a library for valid screening. Thus, we were satisfied with the maximum error in equimolarity (less than 12% for the separated peptides and less than 30% for peaks containing three peptides) of our test peptide obtained using the isokinetic ratios published previously (66).…”

“…The amino acid mixture for a randomized position contained 18 common amino acids (Pro and Cys not included). The mixture was prepared so that the content of each amino acid was in agreement with the isokinetic ratios for competitive Fmocamino acid coupling reported by Kim et al (66). The ratios used were 3.10% (Glu), 3.85% (Gly and Val), 4.60% (Ala, Leu, and Phe), 5.35% (Ile, Met, Gln, His, and Ser), 6.20% (Lys, Thr, and Tyr), 6.95% (Trp), and 7.70% (Asn, Arg, and Asp).…”

“…The isokinetic ratios for Fmoc-amino acids reported earlier (66) were initially examined here to determine whether they were effective for the microwave-assisted SPPS protocols utilized for assembling fTHPs. A short "test" peptide, Xaa-Lys(Dnp)-Ala-Arg-Gly-Lys (where Xaa stands for the mixture of 18 amino acids as listed under "Experimental procedures") was synthesized.…”