MALDI-TOF MS as a label-free approach to rapid inhibitor screening.
Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming 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 <7% 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.
Greis
Kenneth D
KD
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA. greis.kd@pg.com.
Zhou
Songtao
S
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Burt
Thomas M
TM
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Carr
Andrew N
AN
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Dolan
Elizabeth
E
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Easwaran
Vijayasurian
V
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Evdokimov
Artem
A
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Kawamoto
Richard
R
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Roesgen
Jeff
J
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
Davis
Gregory F
GF
Health Care Research Center, Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, 45040, Mason, OH, USA.
D004791:Enzyme Inhibitors / Q000032:analysis* / Q000378:metabolism; D020128:Inhibitory Concentration 50; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D004791:Enzyme Inhibitors; D006899:Mixed Function Oxygenases; D010770:Phosphotransferases