Monitoring enzymatic conversions by mass spectrometry: a critical review

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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confidence: 99%

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confidence: 99%

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

Pei

Kennedy

2010

“…MS offers the significant advantage that it does not require analytes to be labeled, either by direct attachment of fluorescent and radioactive labels or by binding of antibodies, and therefore offers greater flexibility in experiments [12][13][14]. In the past decade, considerable effort was invested to develop MS-based detection schemes capable of assaying inhibition of enzyme-mediated reactions [15,16]. The use of electrospray ionization (ESI)-MS is rapidly expanding into the study of enzyme kinetics [17][18][19][20][21][22][23].…”

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confidence: 99%

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

Yao

Wei

et al. 2008

A matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS)-based assay was developed for kinetic measurements and inhibitor screening of acetylcholinesterase. Here, FTMS coupled to MALDI was applied to quantitative analysis of choline using the ratio of choline/acetylcholine without the use of additional internal standard, which simplified the experiment. The Michaelis constant (K m ) of acetylcholinesterase (AChE) was determined to be 73.9 mol L Ϫ1 by this approach. For Huperzine A, the linear mixed inhibition of AChE reflected the presence of competitive and noncompetitive components. The half maximal inhibitory concentration (IC 50 ) value of galantamine obtained for AChE was 2.39 mol L Ϫ1 . Inhibitory potentials of Rhizoma Coptidis extracts were identified with the present method. In light of the results the referred extracts as a whole showed inhibitory action against AChE. The use of high-resolution FTMS largely eliminated the interference with the determination of ACh and Ch, produced by the low-mass compounds of chemical libraries for inhibitor screening. The excellent correlation with the reported kinetic parameters confirms that the MS-based assay is both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors. The obvious advantages were demonstrated for quantitative analysis and also high-throughput characterization. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for inhibitor screening of

“…These measurements are typically performed by LC/ESI-MS/MS on triple quadrupole instruments to minimize the effects of other contaminating material in the mixtures [1][2][3]. Mass spectrometry techniques have also been widely reported for measuring the conversion of substrates to products for enzyme assays [4]. However, for many enzymes with peptide substrates, ESI-MS approaches have the added complication of dividing the substrate and product into multiple peaks due to charge-state distribution and thus affecting sensitivity and data extraction.…”

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confidence: 99%

MALDI-TOF MS as a label-free approach to rapid inhibitor screening

Greis

Zhou

Burt

et al. 2006

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by timeconsuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC 50 curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were Ͻ7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development. (J Am Soc Mass Spectrom 2006, 17, 815-822)