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

Aminoacyl-tRNA protein transferases catalyze the post-translational 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 sub...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2009-03, Vol.81 (5), p.1937-1943
Main Authors: Ebhardt, H. Alexander, Xu, Zhizhong, Fung, Angela W, Fahlman, Richard P
Format: Article
Language:English
Subjects:
Amino acids
Amino Acids - chemistry
Analytical chemistry
Chemistry
Exact sciences and technology
Isotopes
Mass spectrometry
Peptides
Protein Processing, Post-Translational
Proteins
Spectrometric and optical methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Transfer RNA
Transferases - metabolism
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Summary:Aminoacyl-tRNA protein transferases catalyze the post-translational 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 (MALDI-TOF) 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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac802423d