|Abstract Title:||CHARACTERISATION OF OLIGONUCLEOTIDES BY NEGATIVE ION ESI TANDEM MS AND IMS|
|Presenter Name:||Mr Fabien Hannauer|
|Co-authors:||Prof G. John Langley|
Prof Eugen Stulz
|Company/Organisation:||Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, UK|
Abstract Information :
Introduction The number of therapeutic oligonucleotides approved to the market, such as modified DNA or RNA, that can help for undruggable diseases or diseases with limited treatments such as cancer and rare diseases e.g. Duchenne muscular dystrophy or spinal muscular atrophy has increase since 2016 and have been used recently in vaccines for COVID-19. It is possible to determine the sequence of oligonucleotides, as well as their modifications, by negative ion ESI and tandem mass spectrometry where complex data are obtained which decelerate the drug to be approved for the market. Different software exists to help the identification of the product ions, based on the McLuckey nomenclature, but is limited. Methods Different sequences of 21-mer oligonucleotides were analysed using negative ion ESI using a Waters Synapt G2-Si. Two sample introduction techniques were used, direct infusion and RP-IP-UHPLC with Waters Acquity UHPLC and a C18 BEH column. Triethylammonium acetate and hexafluoroisopropanol in water were used as mobile phase A and triethylammonium acetate in acetonitrile as mobile phase B with a flow rate of 0.25 mL/min and a column temperature at 50°C. Different charge states were isolated within the mass spectrum and fragmented by CID with and without the use of IMS. Preliminary data The use of UHPLC can simplified the spectrum obtained for oligonucleotides by reducing the numbers of adducts present in the sample. Furthermore, to predict how complex oligonucleotides fragment, it is important to understand the fragmentation, mechanisms, and rules of simple oligonucleotides. This will allow to explore mechanisms and determine de novo sequencing, which could be applied for impurities and unknown oligonucleotide sequences. Here, different 21-mer DNA sequences have been characterised to understand the role of internal product ions. Different observations have been obtained depending on the neighbouring of each nucleobase, the nucleobases present at the extremities, and the charge state isolated and fragmented. The different product ions correspond to the McLuckey nomenclature and to internal product ions which helps to understand how simple oligonucleotides fragment and what drives it. This first step will help with the determination of rules and de novo sequencing that could be applied to more complex sequences. IMS, after fragmentation, has been used to allow the separation of the different charges into simplified data, which is well observed for low charge states, and helps to be more confident in the attribution of the different charge states.