Comparison of desorption/ionization on silicon (DIOS) time-of-flight and liquid chromatography/tandem mass spectrometry for assaying enzyme-inhibition reactions.
Desorption/ionization on silicon with time-of-flight mass spectrometry (DIOS-TOFMS) was employed for rapid quantification of small molecules and the measurement of critical constants used in enzyme kinetics studies. The approach was used to determine the Michaelis constant (Km) for acetylcholine esterase and the 50% inhibition constant (IC50) for tacrine. The Km was determined from a Lineweaver-Burk plot to be 98 microM. The IC50 was determined by assaying acetylcholine esterase activity with a range of tacrine concentrations, and measuring the amount of choline produced at a fixed time point by either DIOS-TOFMS or by liquid chromatography tandem mass spectrometry (LC/MS/MS). DIOS-TOFMS indicated an IC50 of 32.0 nM while LC/MS/MS gave a value of 31.8 nM. The excellent correlation with the reported IC50 values, ranging from 30.0 to 50.0 nM, and equivalence with the LC/MS/MS results, confirms that the DIOS-TOFMS method can be used for rapid and specific quantification of enzyme-inhibition assays.
Wall
Daniel B
DB
Waters Corporation, Milford, MA 01757, USA. Daniel_Wall@waters.com
Finch
Jeffrey W
JW
Cohen
Steven A
SA
D000109:Acetylcholine / Q000378:metabolism; D000110:Acetylcholinesterase / Q000378:metabolism*; D002794:Choline / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D012825:Silicon / Q000737:chemistry*; D013619:Tacrine / Q000378:metabolism
D002800:Cholinesterase Inhibitors; D013619:Tacrine; D000110:Acetylcholinesterase; D002794:Choline; D000109:Acetylcholine; D012825:Silicon