Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities.
Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r(2) = 0.9998) over two orders of magnitude of concentration of rac-1-phenylethylamine (PEA), which is converted to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (< or =5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes.
Kang
M J
MJ
Technische Biochemie, Saarland University, Im Stadtwald, Bldg. 2, D-66123 Saarbruecken, Germany.
Tholey
A
A
Heinzle
E
E
D000081:Acetamides / Q000737:chemistry; D000085:Acetates / Q000737:chemistry; D002384:Catalysis; D002849:Chromatography, Gas; D005656:Fungal Proteins; D008049:Lipase / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D010627:Phenethylamines / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
C433670:2-methoxy-N-(1-phenylethyl)acetamide; D000081:Acetamides; D000085:Acetates; D005656:Fungal Proteins; D010627:Phenethylamines; C048765:2-methoxy ethyl acetate; D008049:Lipase; C431349:lipase B, Candida antarctica; C033198:1-phenethylamine