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

• Published in: Analytica chimica acta Vol. 1072; pp. 81 - 86
• Main Authors: Seidl, Claudia, Vilela, Adriana Ferreira Lopes, Lima, Juliana Maria, Leme, Gabriel Mazzi, Cardoso, Carmen Lúcia
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 23-09-2019; Elsevier BV
• Subjects: Acetylcholinesterase; Acetylcholinesterase - analysis; Acetylcholinesterase - metabolism; Assaying; Bioreactors; Butyrylcholinesterase - analysis; Butyrylcholinesterase - metabolism; Choline; Cholinesterase; Cholinesterase inhibitors; Cholinesterase Inhibitors - chemistry; Cholinesterase Inhibitors - pharmacology; Enzyme assay; Enzyme Assays - instrumentation; Enzyme inhibitors; Enzymes; Galantamine; Galantamine - chemistry; Galantamine - pharmacology; Humans; Immobilized enzyme reactor; Ions; Ligand screening; Liquid chromatography; Mass spectrometry; Mass Spectrometry - instrumentation; Mass spectroscopy; Reactors; Scientific imaging; Spectroscopy; Substrates; Ligand screening; Enzyme assay; Immobilized enzyme reactor
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2019.04.057

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.
[Display omitted]
•On-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection.•Two different enzymes are screened at the same time with only one single sample injection and short time analysis.•The system could be adapted to other types of enzyme reactors with different supports.•Enzymes acetylcholinesterase and butyrylcholinesterase were used as proof of concept.