Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities.
Quantitative matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS) was applied for the determination of concentrations of low-molecular-weight (< 400Da) substrates and products of enzyme-catalyzed reactions. Isotope-labeled and fluorinated internal standards were used for the quantification. Automated quantitative MALDI-ToF MS analysis of quenched samples allowed the direct and simultaneous observation of time-dependent decrease of substrate concentration and increase of product concentration without any need for prepurification or desalting steps. The results showed good agreement with established but more elaborate analytical methods. MALDI-ToF MS thus is an interesting alternative tool for the determination of enzyme activities. Due to automated and miniaturized measurement it is especially suitable for the screening of biocatalysts.
Bungert
Ditte
D
Technische Biochemie, Universität des Saarlandes, 66123 Saarbrücken, Germany.
Heinzle
Elmar
E
Tholey
Andreas
A
D043422:Carboxypeptidases A / Q000032:analysis / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation / Q000502:physiology; D005947:Glucose / Q000032:analysis; D005949:Glucose Oxidase / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D005949:Glucose Oxidase; D043422:Carboxypeptidases A; D005947:Glucose