Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

Abstract

Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a highresolution, accurate-mass instrument. We found that in CID spectra acquired under the normalized collision energy (NCE) of 35%, approximately 70% of the total peak intensity was attributed to sequencing ions (a-B, a, b, c, d, w, x, y, z), around 25% of the peak intensity came from precursor ions that experienced complete or partial loss of a nucleobase in the form of either a neutral or an anion, and the remainder were internal ions and anionic nucleobases. The top five sequencing ions were the y, c, w, a-B, and a ions. Furthermore, we observed that CID fragmentation behaviors of RNA oligos were significantly impacted by their precursor charge. Specifically, when the precursors had a charge from 1 − to 5 − , the fractional intensity of sequencing ions decreased, while that of precursors that underwent either neutral or charged losses of a nucleobase increased. Additionally, we found that RNA oligos containing 3′-U tended to produce precursors with HNCO and/or NCO − losses, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings provide valuable insights for better comprehending the mechanism behind RNA fragmentation by MS/MS, thereby facilitating the future automated identification of RNA oligos based on their CID spectra in a more efficient manner.

Effect of terminal phosphate groups on collisional dissociation of RNA oligonucleotide anions

“…The HCD spectra of the two 4-nt RNA oligos (GUCA) with only one terminal phosphate group in turn have a higher peak of PP than those without a terminal phosphate group. The two 6-nt oligos without a terminal phosphate group, 21 although have the same number of phosphate groups as P-GUCA-P, have in their HCD spectra a much lower peak of PP. These data show that the addition of a terminal phosphate group to an RNA oligo, boosts the formation of PP, more than the addition of an internal phosphate group, in the negative-ion HCD.…”

“…For RNA oligos with one or two terminal phosphate groups, the precursors with the loss of phosphoric acid or metaphosphoric acid anion were also calculated and matched with the observed peaks. Cyanate anion (NCO -) loss was not included here since the terminal pyrimidine bases were not contained in all oligos we used 15,30 . The observed monoisotopic peak was assigned to a theoretical ion within a mass tolerance of 10 ppm.…”

“…20 Of all sequencing ions ( a, a-B, b, c, d, w, x, y, z ) resulted from the breaking of the phosphodiester bonds, the five most abundant ones are y, c, w, a-B , and a ions. 21 Factors that affect the fragmentation behaviors of RNA oligos include the precursor charge state, fragmentation energy, metal ion adduction, nucleotide modification, and the presence or absence of 3’-terminal phosphate. 15,17,20…”

“…Mass spectrometry (MS) has become an indispensable analytical method to identify and characterize proteins and small molecules, by virtue of its high sensitivity, high accuracy and high throughput. In recent years, MS has also been increasingly employed in RNA analysis as an expanding field, including RNA sequencing 2,3 , single-nucleotide polymorphism (SNP) identification 4 , post-transcriptional modifications (PTxMs) characterization 5,6 , quality inspection of synthetic RNA 7 , thanks to the comprehensive investigations on MS fragmentation behaviors of nucleic acids during the last 40 years [8][9][10][11][12][13][14][15] .…”

“…The (a-B)-and w-ions, arising from cleavage of 3' C-O bond, which are the primary fragment pathway in CID/HCD spectra of DNA, can also be observed in RNA, but of less abundance [8][9][10] . There are several factors that affect RNA fragmentation behaviors, such as the precursor charge state 12,15,24 , the fragmentation energy deposited 12 , the metal ion adduction 13,24,25 , the modification on nucleotides 11,19 , or phosphates 26 . Our previous study on CID fragmentation patterns of RNA oligos with both terminal hydroxyl groups showed that the sequencing ions (a, a-B, b, c, d, w, x, y, z-type) tend to decrease in overall abundance while the nucleobase losses increased markedly when the precursor charge varies from a low, intermediate to a high state 15 .…”