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MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production

Published online by Cambridge University Press:  24 April 2002

E. JAMES PETERSSON ,
MONA SHAHGHOLI ,
HENRY A. LESTER  and
DENNIS A. DOUGHERTY
E. JAMES PETERSSON
Affiliation:
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
MONA SHAHGHOLI
Affiliation:
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
HENRY A. LESTER
Affiliation:
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA
DENNIS A. DOUGHERTY
Affiliation:
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
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Abstract

Unnatural amino acid mutagenesis requires the in vitro production of aminoacyl tRNAs. Bacteriophage T4 RNA ligase is used to ligate α-amino-protected dCA amino acids to 74mer tRNA. Previously, there has been no facile method for evaluating the efficiency of this reaction prior to using the tRNA in translation. We report a novel use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in monitoring the formation of aminoacyl 76mer tRNA. This method is more efficient and precise than the traditional technique of gel electrophoresis. These MALDI conditions should also prove useful for analyzing aminoacyl tRNAs produced through aminoacyl tRNA synthetases and other methods.

Keywords

aminoacylationgel electrophoresismatrix-assisted laser desorption/ionizationnonsense suppressionT4 RNA ligaseT7 RNA polymeraseTHG73 tRNAunnatural amino acid
Type
METHODS
Information
RNA , Volume 8 , Issue 4 , April 2002 , pp. 542 - 547
Copyright
2002 RNA Society

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