Peptide microarray-based fluorescence assay for simultaneously detecting matrix metalloproteinases

Abstract

A peptide microarray-based fluorescence assay has been proposed for simultaneously determining the activities of MMP-2 and MMP-9 in cell culture medium.

Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality

“…In this proof-of-principle experiment, the interactions of three peptide substrates (S0, S1, and S3) with two MMPs (MMP-2 and MMP-9) were studied. Here, S0 is a control peptide which is not cleaved by MMPs, S1 is a specific substrate of MMP-9, and S3 is a universal substrate of MMP-9 and MMP-2. ,− The enzymatic cleavage reactions were carried out using our previously reported optimum experimental conditions except for the enzymatic cleavage time and peptide substrate concentrations in the spotting solutions . As expected, specific cleavage of peptide substrates by MMPs is clearly observed (as shown in Figure S6 (Supporting Information)).…”

“…For assaying the enzyme activities, the interactions of MMPs (pure MMPs or cell-secreted MMPs) with peptide substrates on the microarrays were evaluated according to our previously reported assay conditions with a slight modification (see the Supporting Information for details) …”

Biosensors and bioassays for determination of matrix metalloproteinases: state of the art and recent advances

“…Besides the above described assays/biosensors, some other techniques have also been applied for quantifying MMPs, such as mass-spectrometry (MS)-based proteomic techniques, 179-182 array/microarray-based assays, [183][184][185][186][187][188] and nuclear imaging approaches. [189][190][191] MS and liquid chromatography-tandem MS (LC-MS/MS) have evolved as promising techniques for qualitative and quantitative analysis of proteins via identification of the unique mass signature of the target.…”

“…Our group has also proposed a series of peptide microarray-based fluorescence arrays for multiplex determination of MMP activities and evaluation of MMP inhibitors. [184][185][186][187][188] In our earlier work, a peptide microarray was fabricated by spotting biotin modified peptide substrates on a commercial chip slide with aldehyde groups. 184 After being incubated with MMP-containing samples, FITC-labeled avidin was applied to the subarrays for fluorescence measurement.…”

“…[184][185][186][187][188] In our earlier work, a peptide microarray was fabricated by spotting biotin modified peptide substrates on a commercial chip slide with aldehyde groups. 184 After being incubated with MMP-containing samples, FITC-labeled avidin was applied to the subarrays for fluorescence measurement. The fluorescence intensities of the spots decreased with increasing MMP activities, and the change of fluorescence intensity (DF) was proportional to the logarithm of the MMP-2 and MMP-9 concentration.…”