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 in epitranscriptomic studies. However, MS fragmentation behaviors of RNA oligos are understood insufficiently. In this study, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos of four to eight nucleotides (nt) in length by collision-induced dissociation (CID) using a high-resolution, accurate-mass instrument. We find that in the CID spectra acquired under the normalized collisional energy of 35%, ~70% of the total peak intensity belonged to sequencing ions (a-B, a, b, c, d, w, x, y, z), ~25% belonged to precursor ions with either complete or partial loss of a nucleobase in the form of a neutral or an anion, and the remainder were internal ions and anionic nucleobases. Of the sequencing ions, the most abundant species were y, c, w, a-B, and a ions. The charge state of the RNA precursor ions strongly affected their fragmentation behaviors. As the precursor charge increased from -1 to -5, the fractional intensity of sequencing ions in the CID spectra decreased, whereas the fractional intensity of precursor ions with neutral and/or charged losses of a nucleobase increased. Moreover, RNA oligos containing U, especially at the 3′ terminus, tended to produce precursors that lost HNCO and/or NCO-, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings build a strong foundation for mechanistic understanding of RNA fragmentation by MS/MS, contributing to future automated identification of RNA oligos from their CID spectra in a more efficient way.

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

“…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.…”

“…Collectively, after CIP treatment, all terminal phosphate groups had been eliminated, and the CID fragmentation behaviors of RNA oligos with both terminal hydroxyl groups we reported in ref. 15 has also indicated that this sample treatment could improve MS analysis for RNA sequencing and PTxM characterization using tandem mass spectrometry. Therefore, we highly recommend CIP treatment prior to MS analysis when the digested products from RNA samples contain either one or two terminal phosphate groups.…”

“…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 .…”

“…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 In this study, we investigated how the 5'-and the 3'-phosphate groups might affect the fragmentation behaviors of RNA oligos in the negative-ion mode.…”