Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry.

Riley Fricke, Isaac Knudson, Cameron Verdayne Swenson, Sarah Smaga, Alanna Schepartz

Journal: Nature protocols 2025;20(5):1246-1274

PMID: 39762443

Full Text on Nature Publishing Group View on PubMed

Abstract

Aminoacyl-tRNA synthetases (aaRSs) provide an essential functional link between an mRNA sequence and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases. In synthetic biology, the acylation of tRNAs with a non-canonical α-amino acid or, more recently, a non-α-amino acid monomer is a critical first step in the incorporation of these monomers into proteins, where they can be used for fundamental and applied science. These endeavors all demand an understanding of aaRS activity and specificity. Here, we describe a liquid chromatography-mass spectrometry assay that directly monitors aaRS activity by detecting the intact acyl-tRNA product. After a simple tRNA acylation reaction workup, acyl- and non-acyl-tRNA molecules are resolved by using ion-pairing reverse-phase chromatography, and their exact masses are determined by using high-resolution time-of-flight mass spectrometry. Our assay is fast and simple, quantifies reaction yields as low as 0.23% and can also be used on tRNAs acylated with flexizyme to detect products that are undetectable by using standard techniques. The protocol requires basic expertise in molecular biology, liquid chromatography-mass spectrometry and RNase-free techniques. This protocol takes ≥5 h to complete, depending on the number of samples.

Address: Department of Chemistry, University of California, Berkeley, CA, USA.; NSF Center for Genetically Encoded Materials (C-GEM), Berkeley, CA, USA.; Department of Chemistry, University of California, Berkeley, CA, USA. [email protected].; NSF Center for Genetically Encoded Materials (C-GEM), Berkeley, CA, USA. [email protected].; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. [email protected].; Chan Zuckerberg Biohub-San Francisco, San Francisco, CA, USA. [email protected].; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA. [email protected].; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. [email protected].; ARC Institute, Palo Alto, CA, USA. [email protected].

DOI: 10.1038/s41596-024-01086-9

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https://doi.org/10.1038/s41596-024-01086-9

MeSH Terms: Chromatography, Liquid, Mass Spectrometry, RNA, Transfer, Transfer RNA Aminoacylation, Amino Acyl-tRNA Synthetases, Acylation, Liquid Chromatography-Mass Spectrometry