Sequence mapping of transfer RNA chemical modifications by liquid chromatography tandem mass spectrometry

Abstract

Mass spectrometry is a powerful analytical tool for identifying and characterizing structural modifications to the four canonical bases in RNA, information that is lost when using techniques such as PCR for RNA analysis. Here we described an updated method for sequence mapping of modified nucleosides in transfer RNA. This modification mapping approach utilizes knowledge of the modified nucleosides present in the sample along with the genome-derived tRNA sequence to readily locate modifications site-specifically in the tRNA sequence. The experimental approach involves isolation of the tRNA of interest followed by separate enzymatic digestion to nucleosides and oligonucleotides. Both samples are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and the data sets are then combined to yield the modification profile of the tRNA. Data analysis is facilitated by the use of unmodified sequence exclusion lists and new developments in software that can automate MS/MS spectral annotation. The method is illustrated using tRNA-Asn isolated from Thermus thermophilus.

Keywords: LC–MS/MS; Modified bases; Modified nucleosides; RNA sequencing; Tandem mass spectrometry; tRNA.

Figure 1

T. thermophiles HB27 tRNA^ASN nucleoside digest. A) Total ion chromatogram, B) Extracted ion chromatogram of the nucleoside N⁶-threonylcarbamoyladenosine, t⁶A m/z 413. C) Tandem mass spectrum of t⁶A showing nucleobase fragment ion (m/z 281.08) as the major ion product. Arrow denotes location of molecular ion, demonstrating complete fragmentation.

Figure 2

Standard nomenclature for annotation of CID fragment ions for oligonucleotides. Figure adapted from [28].

Figure 3

Tandem mass spectrum of RNase T1 digestion product from human placenta ttRNA. MS/MS annotation is consistent with the sequence ACCGp.

Figure 4

Tandem mass spectrum of RNase T1 digestion product from T. thermophilus tRNA^Asn. MS/MS annotation is consistent with the sequence UU[t⁶A]ACCGp. The most abundant peak in the spectrum is the loss of the t⁶A side chain (Table 1).

Figure 5

Tandem mass spectrum of RNase A digestion product from T. thermophilus tRNA^Asn. MS/MS annotation is consistent with the sequence GG[s⁴U]p. Standard modification mapping takes advantage of overlaps to increase sequence coverage and confirm modification placement (Table 2).