DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

Abstract

Abstract. Collision-induced dissociation (CID) of m/z-isolated w type fragment ions and an intact 5′ phosphorylated DNA oligonucleotide generated rearranged product ions. Of the 21 studied w ions of various nucleotide sequences, fragment ion sizes, and charge states, 18 (~86%) generated rearranged product ions upon CID in a Synapt G2-S HDMS (Waters Corporation, Manchester, England, UK) ion mobility-mass spectrometer. Mass spectrometry (MS), ion mobility spectrometry (IMS), and theoretical modeling data suggest that purine bases can attack the free 5′ phosphate group in w type ions and 5′ phosphorylated DNA to generate sequence permuted [phosphopurine] -fragment ions. We propose and discuss a potential mechanism for generation of rearranged [phosphopurine] -and complementary y-B type product ions.

Review of fragmentation of synthetic single‐stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022

“…Those observations were also supported by theoretical calculations using density functional theory at the B3LYP/6-31(d) level of theory and basis set, 88,89 which suggest that the formation of charged PO 3 À and a corresponding neutral purine have lower energy dissociation pathways for [phosphopurines] À than for the formation of neutral HPO 3 and purine ions. The only product ion that cannot produce a phosphopurine is w 1 .…”

“…It remains to be seen whether IMS analysis of product ions derived from different oligonucleotide diastereomers can be used to determine the stereochemistry of the precursor molecule, as has been demonstrated for peptides 86 and oligosaccharides. An internal rearrangement has been observed by Harper et al 88 where they used CID (both off-and on-resonance) and IMS, in negative ESI mode, on isolated w product ions but also intact 5 0 -phosphorylated DNA, corresponding to a w product ion, to obtain rearranged product ions. They concluded that a purine base can attack the free 5 0 -phosphate group, as present in w product ion or 5 0 -phosphorylated for DNA, to generate a rearranged phosphopurine and complementary y-B product ion.…”

Enhancing the Mass Spectrometry Sensitivity for Oligonucleotide Detection by Organic Vapor Assisted Electrospray

“…Oligonucleotides tend to form negative ions in ESI-MS detection due to their backbone phosphodiester groups (p K a < 1), and most of the oligonucleotide analyses including molecular weight determination and structural elucidation were performed in negative mode MS. − Meanwhile, a handful of ESI-MS studies were carried out on positively charged oligonucleotides, − while most authors conceded that negative mode MS was more suitable for oligonucleotide detection with higher sensitivity. Despite the generally accepted benefits of negative mode MS for oligonucleotide detection, being able to detect oligonucleotides in positive mode MS may have some practical advantages.…”

Fast Electron Detachment Dissociation of Oligonucleotides in Electron-Nitrogen Plasma Stored in Magneto Radio-Frequency Ion Traps

“…5,6 However, the dissociation is often too energetic for oligonucleotide precursors produced by electrospray ionization (ESI), i.e., highly deprotonated precursors are crushed into small pieces including internal fragments by secondary fragmentation. 4,7,8 This makes highsensitive sequencing difficult in the middle portion of the oligonucleotides longer than typically 15−20 mers because electrospray ionization produces highly deprotonated precursors mainly.…”

“…The most common technique to dissociate oligonucleotides is collision-induced dissociation (CID) because CID is installed in almost all mass spectrometers for biomolecule analysis. CID, which is induced by thermal excitation of vibrational states, produces a-B and w fragments mainly in the cases of deprotonated DNA , and c and y fragments mainly from deprotonated RNA. , However, the dissociation is often too energetic for oligonucleotide precursors produced by electrospray ionization (ESI), i.e., highly deprotonated precursors are crushed into small pieces including internal fragments by secondary fragmentation. ,, This makes high-sensitive sequencing difficult in the middle portion of the oligonucleotides longer than typically 15–20 mers because electrospray ionization produces highly deprotonated precursors mainly.…”