MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production.
Unnatural amino acid mutagenesis requires the in vitro production of aminoacyl tRNAs. Bacteriophage T4 RNA ligase is used to ligate a-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.
Petersson
E James
EJ
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA
Shahgholi
Mona
M
Lester
Henry A
HA
Dougherty
Dennis A
DA
D000596:Amino Acids / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D012343:RNA, Transfer / Q000032:analysis* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014764:Viral Proteins
D000596:Amino Acids; D014764:Viral Proteins; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases