Detection of protease activities with the mass-spectrometry-assisted enzyme-screening (MES) system

Abstract

The MALDI-MES provides a rapid, sensitive and reproducible alternative approach to existing analytical techniques for the detection of enzymatic activities that does not require a chromophore or radiolabeling. An improved method is presented, by which enzymes with defined substrate specificities can be detected with a MALDI mass spectrometer in complex protein fractions. In order to demonstrate the utility of the new method, in this study we describe the use of MALDI-MES to detect proteolytic activities in a protein extract from porcine renal tissue, which contained several thousand proteins as visualized by 2D electrophoresis. The analytical procedure is based on covalent immobilization of proteins to beads. By immobilizing proteins, autolytic and proteolytic degradation is prevented and the removal of those molecules from the protein fraction is achieved, which otherwise would interfere with the mass spectrometric detection of the enzymatic reaction products. The enzymatic activity is determined by incubating the immobilized proteins with a reaction-specific probe, followed by the analysis of the reaction mixture with the MALDI-MS after defined incubation times. The presence of the target enzyme is validated by locating a signal, which fits the molecular mass of the expected reaction product in the mass spectrum. To demonstrate how to detect proteolytic activities in this system, the reactions catalyzed by endopeptidase, angiotensin-converting enzyme, kallikrein, renin, and urotensin-converting enzyme were monitored. The experiments showed that the MALDI-MES method is sufficient according the quantification to investigate the effects of inhibitors. This is demonstrated using a specific renin inhibitor to inhibit an angiotensin-I generating enzyme activity in a renal protein extract.

International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, Urotensin II–Related Peptide, and Their Receptor: From Structure to Function

“…Porcine kidney tissue exhibits urotensin II-converting enzyme activity as shown by a mass spectrometryassisted enzyme-screening system (Schlüter et al, 2003). Incubation of a 25-amino-acid C-terminal fragment of human pro-UII (CTF-prohUII) with recombinant furin gives rise to a mature 11-amino acid form of UII .…”

New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

“…This is different from spectroscopic assays in which the index of activity is the change in color or fluorescence with no information regarding the identity of the specific peptides formed in the proteolytic reactions. 27 The objective of the present study was to develop a sensitive and specific assay for ACE2 activity. The method was based on our previous work, which established MS as a useful tool for measuring proteolytic enzyme activity, specifically as related to the RAS.…”

Urotensin-II-Converting Enzyme Activity of Furin and Trypsin in Human Cells in Vitro

“…Direct evidence for the expression of a "urotensin-converting enzyme" was demonstrated in porcine renal tissue using a mass spectrometry-assisted enzyme screening system (Schlü ter et al, 2003), although the identity of the convertase involved was not investigated. Multiple high-molecularweight urotensin-II-like peptides were extracted from the superperfusate of cultured SW-13 adrenocortical carcinoma cells, indicating possible proteolytic processing of the prohormone by these cells ).…”