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.

Mass-Encoded Reporters Reporting Proteolytic Activity from within the Extracellular Matrix.

Reporting matrix metalloproteinase (MMP) activity directly from the extracellular matrix (ECM) may provide critical insights to better characterize 2D and 3D cell culture model systems of inflammatory diseases and potentially leverage in vivo diagnosis. In this proof-of-concept study, we designed MMP-sensors, which were covalently linked onto the ECM by co-administration of the activated transglutaminase factor XIIIa (FXIIIa). Elements of the featured MMP-sensors are the D-domain of insulin-like growth factor I (IGF-I) through which co-administered FXIIIa covalently links the sensor to the ECM followed by an MMP sensitive peptide sequence and locally reporting on MMP activity, an isotopically labeled mass tag encoding for protease activity, and an affinity tag facilitating purification from fluids. All sensors come in identical pairs, other than the MMP sensitive peptide sequence, which is synthesized with l-amino acids or d-amino acids, the latter serving as internal standard. As a proof of concept for multiplexing, we successfully profiled two MMP-sensors with different MMP sensitive peptide sequences reporting MMP activity directly from an engineered 3D ECM. Future use may include covalently ECM bound diagnostic depots reporting MMP activity from inflamed tissues.