Quantitation of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Relative peak-height ratios of products to substrates determined by MALDI-TOFMS allow the quantitative analysis of enzyme catalyzed reactions for screening purposes. Two examples were investigated: the first one was a lipase catalyzed reaction which produces 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) using rac-alpha-phenylethylamine (PEA) as substrate. The second one was the pyruvate decarboxylase catalyzed formation of (1R)-1-hydroxy-1-phenyl-2-propanone (PAC) with benzaldehyde (BzA) as substrate. Here the corresponding oximes were analyzed after derivatization using hydroxylamine. The standard curves (r2 = 0.985 for MET, r2 = 0.991 for PAC) were linear over two orders of magnitude for MET and PAC concentrations. After optimization of the sample preparation an average relative standard deviation of 12.5% was obtained in both cases.
Kang
M J
MJ
Technische Biochemie, University of the Saarland, Saarbruecken, Germany.
Tholey
A
A
Heinzle
E
E
D000081:Acetamides / Q000737:chemistry / Q000378:metabolism; D001547:Benzaldehydes / Q000378:metabolism; D002138:Calibration; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000378:metabolism*; D008970:Molecular Weight; D010091:Oximes / Q000032:analysis; D010627:Phenethylamines / Q000378:metabolism; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D000081:Acetamides; D001547:Benzaldehydes; D010091:Oximes; D010627:Phenethylamines; D012997:Solvents; D008049:Lipase; D011767:Pyruvate Decarboxylase; C033198:1-phenethylamine; C032175:benzaldehyde