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Abstract
Amino acid misincorporation during protein synthesis occurs due to misacylation of
tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids
has been observed in a variety of contexts, less work has been done to directly assess
the extent to which specific tRNAs are misacylated in vivo, and the identity of the
misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation
profiling (ISAP), a method to identify and quantify the amino acids attached to a
tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific
isoacceptors tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPhe
and tRNATyr species were isolated from total aminoacyl-tRNA extracted from both yeast
and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable
unbound amino acid and subsequently deacylated. Free amino acids from the deacylated
fraction were then identified and quantified by mass spectrometry. Using ISAP allowed
quantification of the effects of quality control on the accumulation of misacylated
tRNA species under different growth conditions.