Enzyme inhibitor screening by electrospray mass spectrometry with immobilized enzyme on magnetic silica microspheres

Hu, Fengli; Zhang, Huiying; Lin, Huaqing; Deng, Chunhui; Zhang, Xiangmin

doi:10.1016/j.jasms.2008.02.016

Cited by 41 publications

(27 citation statements)

References 45 publications

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“…Assay conditions were modified from a previously reported method [28]. Ten mM NH 4 HCO 3 was used as reaction buffer for all AchE experiments.…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

“…The quenching solvent was found to completely stop the enzymatic reaction and be compatible with MS. NH 4 HCO 3 provided adequate buffering while being compatible with ESI and providing low ion suppression. To improve quantification, chlormequat was included in the quenching solution to act as an internal standard [28]. Typical MS spectra illustrating detection of substrate (acetylcholine), product (choline), and internal standard are shown in Figure 2.…”

Section: Ache Assaymentioning

confidence: 99%

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Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

Pei

Kennedy

2010

J. Am. Soc. Mass Spectrom.

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Electrospray ionization mass spectrometry (ESI-MS) is an attractive analytical tool for high-throughput screening because of its rapid scan time and ability to detect compounds without need for labels. Impediments to the use of ESI-MS for screening have been the relatively large sample consumed and slow sample introduction rates associated with commonly used flow injection analysis. We have previously shown that by segmenting nanoliter plugs of sample with air, an array of discrete samples can be delivered to a platinum-coated emitter tip for ESI-MS analysis with throughput as high as 0.8 Hz and carry-over between samples less than 0.1%. This method was applied to screening for inhibitors of acetylcholinesterase as a demonstration of the potential of segmented flow ESI-MS for such applications. Each enzyme assay consumed 10 nL of sample. At 1 L/min infusion rate, 102 samples were analyzed, corresponding to a 0.65 Hz sample analysis rate. Linear quantification of choline was achieved from 200 M to 10 mM using this method and Z= values were over 0.8 for the assay. Detailed pharmacologic dose-response curves of selected inhibitors were also measured in high-throughput fashion to validate the method. (J Am Soc

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“…Assay conditions were modified from a previously reported method [28]. Ten mM NH 4 HCO 3 was used as reaction buffer for all AchE experiments.…”

Section: Chemicals and Reagentsmentioning

confidence: 99%

Section: Ache Assaymentioning

confidence: 99%

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

Pei

Kennedy

2010

J. Am. Soc. Mass Spectrom.

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“…ACh has been detected by various methods, such as liquid chromatography/electrochemical (LC-EC) method [36][37][38], liquid chromatography/electrospray ionization/ mass spectrometry (LC-ESI-MS) [8,[39][40][41][42], liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC-APCI-MS) [43], as well as MALDI-TOF-MS with CHCA as the matrix [12]. The performances of these methods have been listed in detail on Table 3.…”

Section: Quantitative Methods Validation and Calibration Curvesmentioning

confidence: 99%

“…In this article, a minicompound library was employed to evaluate our new methodology, which contained four known AChE inhibitors (galanthamine, tacrine, Hup A, and Hup B) [8,48] and four control chemical compounds extracted from natural herbs (ferulic acid, rutin, morin, paeonol) with no AChE inhibit effect. Each compound was tested at the molar ratio of 1:1000 (enzyme AChE:each tested compound).…”

Section: Ache Inhibitor Screeningmentioning

confidence: 99%

“…On the other hand, to address the heavy workload of large scale candidate screening, a high-throughput screening methodology is greatly needed. Very recently, mass spectrometry (MS) has been successfully utilized to monitor enzyme reaction [8]. Among which, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) basedtechnology attracted our attention most [9].…”

Section: Introductionmentioning

confidence: 99%

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High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

Liu

et al. 2011

J. Am. Soc. Mass Spectrom.

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In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R 2 =0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R 2 =0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.

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A novel assay to determine acetylcholinesterase activity: The application potential for screening of drugs against Alzheimer's disease

Peng

Rong

Wang

et al. 2017

Biomedical Chromatography

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Acetycholinesterase (AChE) that regulates hydrolysis of acetylcholine (ACh) in the brain, is an important target for treatment of Alzheimer's disease (AD), a feature of which is ACh deficiency. However, the methods to precisely determine AChE activity are still under development. We developed a new method to exploit acetylcholine-d as a surrogate substrate of ACh and measure product choline-d via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay detected activity of AChE present in the normal mouse brain, which is consistent with the standard Ellman assay that determines products spectrophotometrically. In AD mouse models, the result of LC-MS/MS assay showed significant higher AChE activity than that seen in control normal mice, while treatment of AD mice with an AChE inhibitor, huperzine A, led to partial decreases in AChE activity. Our results suggest that this surrogate-based LC-MS/MS method is a new, sensitive and convenient assay for the determination of AChE activity, providing a useful means for screening active compounds that target AChE.

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Enzyme inhibitor screening by electrospray mass spectrometry with immobilized enzyme on magnetic silica microspheres

Cited by 41 publications

References 45 publications

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

Rapid and label-free screening of enzyme inhibitors using segmented flow electrospray ionization mass spectrometry

High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

A novel assay to determine acetylcholinesterase activity: The application potential for screening of drugs against Alzheimer's disease

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