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

Abstract

We report "plasma" electron detachment dissociation (EDD), a novel electron-activated dissociation (EAD) method for the fast sequencing of oligonucleotides with a high sequence coverage. To reduce the repulsive Coulombic force between the deprotonated oligonucleotides and the electron beam, we performed EDD in a neutral electron-nitrogen (N 2 + ) plasma stored in a magneto radio-frequency ion trap. We confirmed that plasma EDD accomplished a high sequence coverage (100%) of RNA with 40 mers in the reaction time of 10 ms using the electron beam kinetic energy of 35 eV. This new technique was applied to various modifications in oligonucleotide therapeutics (ONTs). Phosphorothioate (PS) positions showed an extremely high dissociation efficiency, i.e., 100 times higher than the standard phosphate (PO) in DNA. Locked nucleotides did not show intensive dissociation in EDD; however, collision-induced dissociation (CID) helped sequence these portions. Liquid chromatography− tandem mass spectrometry (LC−MS/MS) using a ZenoTOF mass spectrometer equipped with the plasma EDD technique successfully identified impurities in degraded samples.

Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry

“…The oligonucleotides with PO and PS are commonly detected in negative charge due to the acidic groups, PO and PS. Their sequencing using collision-induced dissociation (CID) with MS/MS (CID-MS/MS) was well studied − because CID produces backbone fragment ions. On the other hand, there are no reports of practical sequence analysis by CID-MS/MS for PMO detected with positive charges.…”

Radical-Induced Dissociation for Oligonucleotide Sequencing by TiO2/ZnAl-Layered Double Oxide-Assisted Laser Desorption/Ionization Mass Spectrometry

“…Although this approach provides higher sequence coverage than previous techniques, a coupled laser is required for the mass spectrometer and may cause safety issues. Furthermore, electron dissociation technologies (ExDs), such as electron capture (ECD), electron transfer (ETD), negative electron transfer (NETD), electron detachment (EDD), electron activated (EAD), and electron photodetachment (EPD) dissociation, also require electron beam excitation (>20 eV) to produce highly enriched product-ion spectra. Nevertheless, electron beams and precursor anions are repulsed by Coulombic forces, limiting the effect of ExDs on the dissociation of the acceptable oligonucleotide length of longer negatively charged oligonucleotides .…”

“…Furthermore, electron dissociation technologies (ExDs), such as electron capture (ECD), electron transfer (ETD), negative electron transfer (NETD), electron detachment (EDD), electron activated (EAD), and electron photodetachment (EPD) dissociation, also require electron beam excitation (>20 eV) to produce highly enriched product-ion spectra. Nevertheless, electron beams and precursor anions are repulsed by Coulombic forces, limiting the effect of ExDs on the dissociation of the acceptable oligonucleotide length of longer negatively charged oligonucleotides . The recently developed radical transfer dissociation (RTD) technique utilizes the ·H radical to activate oligonucleotides up to 39-mer .…”

“…Nevertheless, electron beams and precursor anions are repulsed by Coulombic forces, limiting the effect of ExDs on the dissociation of the acceptable oligonucleotide length of longer negatively charged oligonucleotides. 25 The recently developed radical transfer dissociation (RTD) technique utilizes the •H radical to activate oligonucleotides up to 39-mer. 26 However, multiple charge states of the oligonucleotide ions are generated upon electrospray ionization (ESI), which disperses the signal intensity when the precursor ion is used in tandem mass spectrometry, leading to overall low signal intensities in the MS/MS spectra.…”

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

“…They suggest that this 'new dissociation technique, RTD, allows for de novo sequence characterisation of modified RNA without the need for laborious sample preparation or specialised MS instrumentation'. It could be interesting to apply this technique to a wider range of modifications to RNA as well as to DNA and to compare the different sequence coverages to those of CID to determine its advantages and limitations.The second new technique, 'plasma' EDD, which is an electronactivated dissociation (EAD) method, was developed by Karasawa et al80 for fast sequencing of oligonucleotides. Plasma EDD is performed in a neutral electron-nitrogen plasma stored in a magneto radio-frequency ion trap using an electron beam kinetic energy of approximately 25-35 eV for 10-20 ms.…”