Journal: Journal of Medicinal Chemistry

Article Title: Development of Selective ADAMTS-5 Peptide Substrates to Monitor Proteinase Activity

doi: 10.1021/acs.jmedchem.2c02090

Figure Lengend Snippet: Design and screening of an ADAMTS-5 combinatorial substrate library and identification of peptide hits. (A) Design of an ADAMTS-5 combinatorial library. The FRET peptide substrate KY(NO 2 )TESESRGK(Abz)IYYKKG ( 3 ) was used as the basis for the library, with fluorophore and quencher positions denoted in red and blue, respectively. Amino acid changes at specific positions are shown by their single letter code, except unnatural amino acids (shaded in purple) which are abbreviated as follows: O = ornithine, B = 2,4-diaminobutyric acid, J = β-cyclopropyl-alanine, Z = 4-thiazolyl-alanine, U = 2,3-diaminopropionic acid. (B) Fluorescent images of library beads prior to screening with TS5-5 (0 h), after screening with TS5-5 (0.5 μM, 30 min, 1 h) and after deactivation with TFA (24 h) in TNC buffer with 0.005% (v/v) Brij-35. Beads were imaged under a fluorescent microscope (λ ex = 350 nm and λ em = 438 nm) and images processed and false colored using Fiji software. (C) Example mass spectrum of a peptide hit identified from ADAMTS-5 combinatorial library screening. Tandem mass spectrum following fragmentation of [M – O + H] + (2023.00523 u), where M = mass of the quasi-molecular ion. For clarity, only selected residue losses are shown and only fragment ion peaks that could be identified as b ions or y ions have been labeled. The spectrum was analyzed and processed using Bruker Compass DataAnalysis software.

Article Snippet: Bruker Compass DataAnalysis was used to analyze both MALDI and tandem mass spectra, while LibMSCalc was used to score each peptide hit.

Techniques: Microscopy, Software, Library Screening, Residue, Labeling