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-T OFMS allow the quantitative analysis of enzyme catalyzed reactions for scre ening 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-phe nyl-2-propanone (PAC) with benzaldehyde (BzA) as substrate. Here the corres ponding oximes were analyzed after derivatization using hydroxylamine. The standard curves (r(2) = 0.985 for MET, r(2) = 0.991 for PAC) were linear ov er two orders of magnitude for MET and PAC concentrations. After optimizati on of the sample preparation an average relative standard deviation of 12.5 % was obtained in both cases. Copyright (C) 2000 John whey & Sons, Ltd.