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

Abstract

The fragmentation of oligonucleotides by mass spectrometry allows for the determination of their sequences. It is necessary to understand how oligonucleotides dissociate in the gas phase, which allows interpretation of data to obtain sequence information. Since 2014, a range of fragmentation mechanisms, including a novel internal rearrangement, have been proposed using different ion dissociation techniques. The recent publications have focused on the fragmentation of modified oligonucleotides such as locked nucleic acids, modified nucleobases (methylated, spacer, nebularine and aminopurine) and modification to the carbon 2'-position on the sugar ring; these modified oligonucleotides are of great interest as therapeutics. Comparisons of different dissociation techniques have been reported, including novel approaches such as plasma electron detachment dissociation and radical transfer dissociation. This review covers the period 2014-2022 and details the new knowledge gained with respect to oligonucleotide dissociation using tandem mass spectrometry (without priori sample digestion) during that time, with a specific focus on synthetic single-stranded oligonucleotides.

FIGURE 1

Structures of nucleobases and oligonucleotide. Reproduced from Hannauer et al.

FIGURE 2

From left to right: structures of LNA, PNA and morpholino phosphoroamidate.

FIGURE 3

McLuckey nomenclature for oligonucleotide fragmentation.

FIGURE 4

Fragmentation pathway for the formation of a‐B and w in DNA sequences. Adapted from Wan et al.

FIGURE 5

Dissociation pathway for the formation of c and y in RNA sequence with (A, B) two different mechanisms. Adapted from Tromp and Schürch.

FIGURE 6

Structures of different modified nucleosides. Y: 2‐aminopurine; D: 2′‐deoxyribose spacer; R: ribose spacer; N: nebularine; a′: 2′‐OCHE adenosine; P: propanediol linker. Analysed in Riml et al.

FIGURE 7

Proposed mechanism for phosphodiester backbone bond cleavage for [M + nH] ⁿ⁺ ions of RNA in CID (off‐resonance). Reproduced from Fuchs et al.

FIGURE 8

(A) Proposed mechanism for the release of NCO⁻ upon CID (on‐resonance) of highly negatively charged oligonucleotide. (B) Postulated mechanism for metaphosphate excision as subsequent reaction to NCO⁻ and water loss. Reproduced from Nyakas et al.

FIGURE 9

Structure of homo‐DNA.

FIGURE 10

Structures of the methylcytidine, methyladenosine, methylguanosine, methyluridine and thiouridine monophosphate isomers analysed in Jora et al. and Li et al.

FIGURE 11

Proposed CID (on‐resonance) pathways for (A) methyladenosine, (B–D) methylguanosine, (E) methylcytidine and (F) methyluridine adapted from Li et al. Original assignment of loss of NH replaced with re‐interpretation as loss of ^•CH₃ as denoted by *, with consequent new proposals of product ion structures.

FIGURE 12

Proposed dissociation products of ETD in positive ion ESI where w or d ion carries a negative charge and the complementary product ion harbours a positive charge. Reproduced from Hari et al.

FIGURE 13

Proposed mechanism for the formation of the product ions d and w by RTD. Reproduced from Calderisi et al.

FIGURE 14

Proposed mechanism for the generation of rearranged [phosphopurine]⁻ and y‐B ions. Adapted from Harper et al.