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

Zhen Lei^1,2, Zhenxin Wang^3,4

¹School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, China.

Methods in Molecular Biology (Clifton, N.J.)

|September 24, 2022

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This study introduces a peptide array assay for profiling enzyme activity, specifically matrix metalloproteinases (MMPs). The method accurately identifies MMP expression and activity in cells and tissues without prior separation.

Area of Science:

Biochemistry
Molecular Biology
Assay Development

Background:

Enzyme activity profiling is crucial for understanding biological processes.
Matrix metalloproteinases (MMPs) play significant roles in various physiological and pathological conditions.
Existing methods for MMP activity determination can be complex and lack spatial resolution.

Purpose of the Study:

To develop and validate a peptide array-based in situ fluorescence assay for high-throughput profiling and localization of MMP activity.
To assess the sensitivity and applicability of the assay in cell monolayers and tissue sections.
To evaluate the potential of the assay for drug discovery by assessing compound inhibition.

Main Methods:

Fabrication of peptide arrays with specific MMP substrates (MMP-2, -3, -7, -9, -14) featuring a FRET pair (FAM/Dabcyl).

Keywords:

High-throughput analysis In situ fluorescence Matrix metalloproteinases (MMP)Peptide array Tissue section

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Application of the peptide array to cell monolayers and thyroid tissue sections for in situ analysis.

Detection of MMP activity via fluorescence intensity resulting from internalized hydrolyzed peptide fragments.

Evaluation of the inhibition of MMP activity by six different compounds.

Main Results:

The assay successfully profiled MMP expression patterns in cells and determined MMP activities in cell monolayers with high sensitivity (down to hundreds of cells/cm²).
Localized MMP activities in 32 thyroid tissue samples were accurately profiled without the need for separation or extraction.
The inhibition potencies of six compounds against five MMPs were successfully evaluated.

Conclusions:

The peptide array-based in situ fluorescence assay is a reliable and efficient tool for high-throughput MMP activity profiling and localization.
This method offers high sensitivity and spatial resolution, making it valuable for biological research and drug discovery.
The assay's applicability to tissue sections demonstrates its potential for clinical diagnostics and biomarker discovery.