Enzyme Inhibitor Screening by Electrospray Mass Spectrometry with Immobilized Enzyme on Magnetic Silica Microspheres

In this study, a novel technique for screening inhibitors by electrospray mass spectrometry (ESI-MS) with immobilized enzyme on magnetic microspheres has been demonstrated. First, the model enzyme acetylcholinesterase (AChE) is immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic silica microspheres. AChE activity was monitored by biochemical assay that is based on mixing of AChE immobilized microspheres and model substrate acetylcholine, separating and detecting the product through ESI-MS. Stability of the enzyme-immobilized microspheres was investigated. No apparent loss of enzyme activity was observed after fivefold reuse of AChE-immobilized microspheres. The enzyme-immobilized bioassay was used to effectively identify AChE inhibitors among two standard samples, huperzine A and huperzine B, and their source herbal Huperzia serrata, all of which were spiked into the substrate. The inhibition was determined by measuring a decrease of product formation using ESI-MS.