Peptide Array-Based In Situ Fluorescence Assay for Profiling Multiple Matrix Metalloproteinase Activities.

Abstract

Peptide array-based in situ fluorescence assay is a reliable and efficient technique for high-throughput profiling and localization of enzyme activity. Here, peptide array is fabricated by spotting five specific MMPs (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14) peptide substrates containing FAM/Dabcyl fluorescent resonance energy transfer (FRET) pair on the surface of cell monolayers or tissue sections. MMP activities are determined in situ by the fluorescence intensity of stained cells/tissues due to the cellular internalization of hydrolyzed peptide fragments with FAM moieties. Identification of MMP expression patterns of cells, highly sensitive determination of MMP activities in cell monolayer (as low as hundreds of cells per square centimeter), and evaluation of inhibition potencies of six compounds toward five MMPs are achieved by this method. Five MMP activities in the localized parts of 32 thyroid tissues are also well profiled without separation or extraction procedures.