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

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.

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.

Interplay between chromophore binding and domain assembly by the B12-dependent photoreceptor protein, CarH.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here - using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy - we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications.

Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS.

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.