A Complete and Versatile Protocol: Decoration of Cell-Derived Matrices with Mass-Encoded Peptides for Multiplexed Protease Activity Detection.

This article provides guidance toward a platform technology for monitoring enzyme activity within the extracellular matrix (ECM) assessed by quantifying reporters secreted into the cell culture supernatant and analyzed by tandem mass spectrometry. The reporters are enzymatically and covalently bound to the ECM by transglutaminases (TG) using the peptide sequence of human insulin-like growth factor I's (IGF-I) D-domain which is known to be bound to the ECM by transglutaminase. The IGF-I D-domain sequence is followed by a peptide sequence cleaved by the intended target protease. This protease-sensitive peptide sequence (PSS) is cleaved off the ECM and can be used to monitor target-enzyme activity by employing a downstream mass tag designed according to isobaric mass encoding strategies, i.e., the combination of isotopically labeled, heavy amino acids. Thereby, cleavage events are linked to the appearance of encoded mass tags, readily allowing multiplexing. This article presents the design and synthesis of these mass reporters. It further aims at detailing the search for peptide sequences responding to target proteases to facilitate future work on enzyme activity measurement for enzymatic activities of hitherto unknown enzymes. In conclusion, the goal of this article is to arm scientists interested in measurements of local enzymatic activities within the ECM with robust protocols and background knowledge.