Mapping Post-Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

Abstract

Liquid chromatography, coupled with tandem mass spectrometry, has become one of the most popular methods for the analysis of post-transcriptionally modified transfer ribonucleic acids (tRNAs). Given that the information collected using this platform is entirely determined by the mass of the analyte, it has proven to be the gold standard for accurately assigning nucleobases to the sequence. For the past few decades many labs have worked to improve the analysis, contiguous to instrumentation manufacturers developing faster and more sensitive instruments. With biological discoveries relating to ribonucleic acid happening more frequently, mass spectrometry has been invaluable in helping to understand what is happening at the molecular level. Here we present a brief overview of the methods that have been developed and refined for the analysis of modified tRNAs by liquid chromatography tandem mass spectrometry.

Keywords: modified nucleosides; LC‐MS/MS; RNA sequencing; tRNA; tandem mass spectrometry.

Figure 1

Examples of different types of RNA modifications. (A) 1-methylguanosine (m¹G) methylation; (B) pseudouridine (ψ) isomerization; (C) 5-formylcytidine (f⁵C) formylation; and (D) wybutosine (yW) a hypermodification as the result of multiple enzymatic reactions.

Figure 2

Schematic of electrospray process. Adapted from [32].

Figure 3

Sequence ladder of possible fragmentation sites for an RNA oligonucleotide by collision-induced dissociation fragmentation. B₁, B₂, etc. represent the nucleobase of the RNA, while a₁, w₁, etc. are the accepted nomenclature to denote the bond that is cleaved. Adapted from [41].

Figure 4

Flowchart of RNA modification mapping experiment using enzymatic digestion for generation of both nucleoside and oligonucleotide samples to be analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). SVP: snake venom phosphodiesterase; BAP: bacterial alkaline phosphatase; RNase: ribonuclease.

Figure 5

Representative example of RNA modification mapping by LC-MS/MS. (a) Total ion chromatogram (TIC) and extracted ion chromatogram (XIC) for RNase T1 digest of Lactococcus lactis. Only one peak for the XIC of m/z 807 is detected; (b) This doubly-charged m/z value is consistent with a digestion product of composition (U₃CGp + 30). Two known modified nucleosides have a mass change of 30 Da: 5-methoxyuridine (mo⁵U) and 5-methyl-2-thiouridine (m⁵s²U). As only the former was found in the nucleoside digest, mo⁵U is most likely the modified nucleoside in this digestion product; (c) Tandem mass spectrometry (MS/MS) data confirming sequence as CUU[mo⁵U]Gp, which maps onto Ala-tRNA^UGC. Figure adapted from [58] with permission.

Figure 6

Schematic outline of comparative sequencing of total transfer ribonucleic acids (tRNAs) from an unknown (candidate) organism using a known (reference) organism by isotope-labeling and LC-MS. tRNA endonuclease digestion products that are equivalent between organisms will appear as doublets (separated by 2 Da) in the mass spectral data; digestion products that are different between the two organisms will appear as a singlet. Figure adapted from [67] with permission.