Global identification of transfer RNAs by liquid chromatography–mass spectrometry (LC–MS)

Transfer ribonucleic acid (tRNA) is the non-coding RNA that links the processes of gene transcription with protein translation. While tRNAs have, individually, been studied for many years, few approaches exist for the global identification of tRNAs at the RNA and posttranscriptional RNA levels. Previously our lab introduced the concept of signature enzymatic digestion products (SDPs) for tRNA identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). SDPs enable the direct determination of tRNA identity based on mass spectrometry detection of unique m/z values from enzymatic digestion products. Here we have examined the applicability of liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) for global identification of bacterial tRNAs via their SDPs using Escherichia coli as the model system. Optimal ultra high performance and high performance liquid chromatography (UPLC vs. HPLC) conditions were identified to address the hundreds of enzymatic digestion products present in the sample. The use of LC–MS/MS improves the accuracy of SDP assignments through confirming sequence information. The combination of mass unique SDP detection along with MS/MS sequencing yielded the identification of all tRNA families from E. coli and nearly doubles the number of specific SDPs detected over that previously obtained using MALDI-MS. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

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► A method for direct identification of bacterial transfer RNA is presented.
► Liquid chromatography–mass spectrometry yields improved detection of tRNAs.
► LC–MS with LC–MS/MS improves confidence in tRNA identifications.
► This LC–MS(/MS) method should be useful for archaeal and eukaryal tRNAs.