Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis

Abstract

Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid chromatography coupled with high-resolution tandem mass spectrometry (HRMS/MS) have resulted in efficient methods for qualitative and quantitative adductomics. We developed an HRMS-based method for the simultaneous analysis of RNA and DNA adducts in a single run and demonstrated its application using Baltic amphipods, useful sentinels of environmental disturbances, as test organisms. The novelty of this method is screening for RNA and DNA adducts by a single injection on an Orbitrap HRMS instrument using full scan and data-independent acquisition. The MS raw files were processed with an open-source program, nLossFinder, to identify and distinguish RNA and DNA adducts based on the characteristic neutral loss of ribonucleosides and 2'-deoxyribonucleosides, respectively. In the amphipods, in addition to the nearly 150 putative DNA adducts characterized earlier, we detected 60 putative RNA adducts. For the structural identification of the detected RNA adducts, the MODOMICS database was used. The identified RNA adducts included simple mono- and dimethylation and other larger functional groups on different ribonucleosides and deaminated product inosine. However, 54 of these RNA adducts are not yet structurally identified, and further work on their characterization may uncover new layers of information related to the transcriptome and help understand their biological significance. Considering the susceptibility of nucleic acids to environmental factors, including pollutants, the developed multi-adductomics methodology with further advancement has the potential to provide biomarkers for diagnostics of pollution effects in biota.

Figure 1

DNA- and RNA-methylation catalyzed by DNA methyltransferases and RNA methyltransferases, respectively, using S-adenosylmethionine as the methyl donor.

Figure 2

EIC using m/z [M + H]⁺ (± 5 ppm) of each 2′-deoxyribonucleosides (A) and ribonucleosides (B). In (A), panel 1 for m/z 268.1040, corresponding to the molecular ion for dG, showed two peaks. Inset shows EIC of nucleobase fragments, guanine (m/z 152.0567) and adenine (m/z 136.0618) from the same DIA window range of m/z 250–270, which demonstrate that the peak at 6.5 min in panel 1 corresponds to dG, whereas the peak at 4.4 min in the same panel corresponds to Ado (also shown in B, panel 2). In both (A,B), all peak assignments were confirmed by comparison with respective standards.

Figure 3

RNA adductome map based on the detected adducts in amphipods. Each circle represents an individual ribonucleoside adduct, with retention time plotted on the X-axis and m/z of the precursor ion on the Y-axis, and the circle size corresponding to relative abundance of each adduct (based on the EIC peak area of the adduct and normalized to that of Guo, adduct area × 10²/Guo area). All structurally identified adducts are shown in respective colors.

Figure 4

Identification of Ino, 5-me-Cyt, 7-me-Guo, N⁶-me-Ado, and 5-me-Urd in the amphipod RNA using respective standards. An overlap of the EIC peaks, using m/z of respective [M + H]⁺ (±5 ppm), corresponding to the ribonucleoside adducts in amphipod samples before (solid line) and after (dotted line) spiking with the respective standards to confirm the identification. EIC for m/z 259.0925 in amphipod showed several peaks, but only the third peak (retention time 4.9 min) had a relative increase in intensity after spiking the standard, indicating that this peak corresponds to 5-me-Urd. The peaks marked with an asterisk (*) are not affected by the spiking, suggesting that they might be potential isomers in EIC for 5me-Urd (which also had the fragment nucleobase adduct ion detected) or minor impurities in EICs for 5-me-Cyt, N⁶-me-Ado, and 7-me-Guo (for which the fragment corresponding to the respective nucleobase adduct ions was not detected).

Figure 5

Representation of nucleic acid cleavage (A) and selective detection (B) of RNA- and DNA-nucleoside adducts. After hydrolysis of DNA and RNA, the nucleosides are characterized by three parts: the base, the modifier part, and the sugar ring deoxyribose (dR from DNA) or ribose (R from RNA). Detection, using HRMS, is based on the identification of the two ions—parent (molecular) ion and specific fragment (nucleobase adduct) ion—and the neutral loss that differs between DNA (dR, 116.0473 Da) and RNA (R, 132.0423 Da).