Quantification of the Post-Translational Addition of Amino Acids to Proteins by MALDI-TOF Mass Spectrometry

摘要

Aminoacyl-tRNA protein transferases catalyze the posttranslational addition of amino acids to proteins. The eubacterial leucyl/phenylalanyl-tRNA-protein transferase (L/F transferase) catalyzes the transfer of leucine or phenylalanine from their respective aminoacylated tRNAs to the N-termini of substrate proteins possessing an N-terminal lysine or arginine amino acid. Conventional assays to quantify L/F transferase activity involve measuring radioactive amino acid incorporation into substrate proteins. We have developed a quantitative matrix assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry procedure to measure the enzymatic activity of L/F transferase. The procedure utilizes stable isotope labeled substrate and internal standard peptides. The method is used to determine the kinetic parameters of k_(cat) and K_(m) for the enzymatic transfer of phenylalanine and three unnatural amino acid derivatives from an aminoacyl-tRNA to a peptide substrate.