Mass-Encoded Suspension Array for Multiplex Detection\nof Matrix Metalloproteinase Activities

Abstract

This\nwork designed a mass spectrometric biosensing strategy for\nthe multiplex detection of matrix metalloproteinases (MMPs) with a\nmass-encoded suspension array. This array was fabricated as multiplex\nsensing probes by functionalizing magnetic beads with MMP-specific\npeptide-isobaric tags for relative and absolute quantification (iTRAQ)\nconjugates, which contained a hexahistidine tag for surface binding,\na substrate region for MMP cleavage, and a coding region for the specific\nMMP. The integration of the multiplex coding ability of iTRAQ with\nultrahigh performance liquid chromatography-tandem mass spectrometry\n(UPLC-MS/MS) and the proteolysis method for peptide digestion endowed\nthe biosensing method with high throughput and ultrahigh sensitivity.\nThis strategy could be conveniently performed by mixing the sample\nand the suspension array for enzymatic reactions and then digesting\nthe uncleaved peptides with trypsin to release the coding regions\nfor UPLC-MS/MS analysis. With MMP-2 and MMP-7 as analytes, the relative\nchanges of peak area ratios of coding regions showed good linear responses\nin the ranges of 0.2–100 and 0.5–400 ng mL^–1, with detection limits of 0.064 and 0.17 ng mL^–1, respectively. The analysis of MMP activity in serum samples and\nits change responding to inhibitors demonstrated the specificity,\npracticability, and expansibility of the proposed strategy. This work\npaves a new avenue for the activity assays of multiplex enzymes and\npromotes the development of mass spectrometric biosensing.