We describe a new assay that reports directly on the acylation state of a user-chosen transfer RNA (tRNA) in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non-α-amino acid monomers into protein: (i) the origins of the apparent selectivity of translation with respect to β2-hydroxy acid enantiomers; (ii) the activity of PylRS variants for benzyl derivatives of malonic acid; and (iii) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2',3'-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non-α-amino acid monomers into proteins.
There is enormous interest in generating proteins composed of monomers that are not ⍺-amino acids. Doing so in cells demands high levels of precisely acylated transfer RNA (tRNA) molecules. However it is exceptionally challenging to simultaneously determine precisely how much of a cellular tRNA is acylated, what it is acylated with, and whether it has been acylated once or twice. The assay presented in this paper, called PARTI, provides exactly this information. It relies on high-resolution mass spectrometry to identify the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. The paper describes the development and validation of the assay and its application to understand three recent observations related to the cellular incorporation of non-⍺-amino acid monomers into protein: (i) the origins of the apparent selectivity of translation with respect to β2-hydroxy acid enantiomers (reported in Hamlish et al. 2024); (ii) the activity of a PylRS variant for benzyl derivatives of malonic acid (reported in Fricke et al. 2023); and (iii) the apparent inability of certain N-Me amino acids to function as ribosome substrates in living cells.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.