Assessing protease activity pattern by means of multiple substrate ESI-MS assays.

Area of Science:

• Biochemistry and Analytical Chemistry
• Enzymology and Proteomics

Background:

• Traditional enzyme activity assays are often limited to single substrate analysis.
• High-throughput screening of enzyme activity requires efficient and multiplexed methodologies.

Purpose of the Study:

• To develop and validate a simultaneous multiple substrate enzymatic assay.
• To apply this assay for parallel screening of proteolytic enzyme activities.
• To compare MS-based assay results with conventional UV/vis detection methods.

Main Methods:

• Development of a multiplexing assay using electrospray ionization mass spectrometry (ESI-MS).
• Assessment of six proteolytic enzymes (trypsin, thrombin, chymotrypsin, bromelain, ficin, elastase) against seven distinct substrates.
• Semi-quantitative evaluation of enzyme activity patterns using a five-class ranking system.

Main Results:

• The ESI-MS multiplexing assay successfully screened activities of multiple enzymes against various substrates simultaneously.
• Activity patterns were generated and classified, showing good agreement with single substrate UV/vis assays.
• The method demonstrated reliability in assessing enzyme specificity and activity levels.

Conclusions:

• The developed ESI-MS-based multiplexing assay is a valid and efficient tool for parallel proteolytic enzyme activity screening.
• This approach offers a robust alternative to conventional single-substrate assays, enabling comprehensive enzyme characterization.
• The assay facilitates semi-quantitative evaluation of enzyme-substrate interactions, aiding in enzyme discovery and development.