A novel on-flow mass spectrometry-based dual enzyme assay

Area of Science:

• Analytical Chemistry
• Biochemistry
• Enzyme Assays

Background:

• Enzyme activity monitoring is crucial for drug discovery and biochemical research.
• Traditional enzyme assays can be time-consuming and require multiple steps.
• Developing high-throughput, simultaneous enzyme screening methods is essential.

Purpose of the Study:

• To develop a novel simultaneous on-flow dual parallel enzyme assay.
• To enable rapid, label-free screening of enzyme inhibitors.
• To demonstrate the system's adaptability and efficiency.

Main Methods:

• Utilized immobilized enzyme reactors (ICERs) in a parallel configuration.
• Integrated liquid chromatography (LC) with mass spectrometry (MS) detection.
• Employed two 10-port/two-position switching valves for system control.
• Monitored enzymatic hydrolysis products of acetylcholine (ACh) using mass spectrometry.

Main Results:

• Achieved simultaneous monitoring of two different enzymes (AChE and BChE) in a single run.
• Assay completion time was less than 6 minutes per sample.
• Demonstrated independent monitoring of each enzyme's activity.
• Successfully screened a cholinesterase inhibitor (galantamine) in a label-free manner.

Conclusions:

• The developed dual parallel enzyme assay is a fast and efficient tool for simultaneous enzyme activity monitoring.
• This method facilitates rapid, label-free screening of enzyme inhibitors.
• The system's design offers flexibility for adapting different enzyme reactors and supports.