Investigation of the Influence of Charge State and Collision Energy on Oligonucleotide Fragmentation by Tandem Mass Spectrometry

Abstract

Due to the increasing pharmaceutical interest of oligonucleotides, for example in antisense therapy and vaccines, their analytical characterization is of fundamental importance due to their complex structure. For this purpose, mass spectrometry is a viable tool for structural studies of nucleic acids. Structural information regarding the primary sequence of a nucleic acid can reliably be gained via tandem mass spectrometry (MSMS) fragmentation. In this work, we present the characteristic fragmentation behavior of short-chain oligonucleotides (15–35 nucleotides) with respect to the collision-induced dissociation (CID) voltage used. The relationship and influence of the length of the oligonucleotide and its charge state is also discussed. The results presented here can be helpful for estimating the required fragmentation energies of short-chain oligonucleotides and their sequencing.

Integrating Internal Fragments in the Interpretation of Top-Down Sequencing Data of Larger Oligonucleotides

“…In contrast, higher charge states up to 15- exhibited more terminal fragments at lower KE values, while the internal ones predominated at higher KE (Figure d–e). Overall, these results corroborated prior observations that low activation energy tends to favor the production of informative terminal fragments by the more highly charged precursor ions. ,, …”

“…Overall, these results corroborated prior observations that low activation energy tends to favor the production of informative terminal fragments by the more highly charged precursor ions. 12,20,27 It has been hypothesized that the divergent behavior of lower charge states might stem from lower intramolecular repulsion between like charges, which can affect the ability to observe fragmentation products in different ways, including through the putative preservation of a higher order structure in the gas phase. In this direction, it has been shown that the intramolecular interactions that stabilize the NA secondary structure are capable of cumulatively curbing the observation of classic terminal fragments by preventing the full dissociation of base-paired sections.…”

Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime

“…In this experiment, to model conventional conditions, we have used Ar collision gas and 5 × 10 −3 mbar pressure indicated on the ion gauge, as suggested by the manufacturer. The result is shown in Figure 1 a (yellow points), analogous curves have often been published [ 7 , 13 , 22 , 25 , 28 ]. This shows that increasing the (laboratory frame) collision energy to ca.…”

“…Increasing the collision energy increases the internal energy of the colliding ions, and that in turn increases the degree of fragmentation, changes the ratio of various fragment ion abundances, and may also change fragmentation routes. Varying and optimizing the collision energy has various practical applications, such as determination of binding energies [ 8 , 9 ], differentiation of isomers [ 10 , 11 , 12 ], characterization of oligonucleotides [ 13 ] and small molecules [ 14 , 15 , 16 , 17 ], and various proteomic applications [ 18 , 19 , 20 ] including quantitation [ 21 ].…”