High-Resolution Ion Mobility-Mass Spectrometry of Isobaric Ribonucleotides Variants
Abstract 294943
Reza Nemati1; Andrew Baker2; Jakub Ujma3; Christopher DeMott4; Lucas Davison1; Kathleen McDonough4; Maksim Royzen1; Daniele Fabris1
1University at Albany, Albany, NY; 2Waters Corporation, Pleasanton, California; 3Waters Corporation, Wilmslow, United Kingdom; 4Wadsworth Center/New York State Department Of Health, Albany, NY
Introduction
Over 140 post-transcriptional modifications (PTM) decorate natural RNA, which fine-tune the RNA structure and function. High-throughput analysis of RNA PTMs has been hindered by the lack of analytic tools for the identification and quantification of isobaric ribonucleotide variants. We explored the possible utilization of high-resolution ion mobility spectrometry (IMS) MS to achieve this task. We employed a novel cyclic IMS cell embedded in a Q-TOF instrument, which significantly increases resolution by extending the delay time between ion injection and ejection from the cyclic mobility separator. The results demonstrated the ability to discriminate between species with the same elemental composition but different structures, thus demonstrating the potential of this high-resolution technique for the analysis of complex isobaric mixtures.
Methods
Mixed ribonucleotide monophosphates and cyclic ribonucleotide monophosphate (cNMP) isomers were either purchased or synthesized in-house. Sample solutions were infused into a Synapt G2 HDMS and a prototype Q-TOF, which was fitted with a one-meter effective path length cyclic ion mobility separator operated with nitrogen as the mobility gas. Experiments were performed in positive and negative ion mode. IMS pathlength and resulting IMS resolution were increased by increasing residence time in the cyclic IMS separator. MS/MS spectra of mobility resolved analytes were generated by elevating the voltage applied to a traveling wave ion guide placed before the TOF entrance.
Preliminary Data
Among the over 140 known variants, around 60 of them are position isomers in which a post-transcriptional modifier is placed on different positions of a canonical ribonucleotide. For this reason, these species share the same elemental composition and thus mass. In most cases, isomers can be discriminated from one another by tandem MS, which can provide unique fragments or diagnostic transitions upon multiple activation steps (i.e., MSn). However, position isomers can be also discriminated according to their IMS behavior, which is a result of their unique conformations. We, therefore, evaluated the virtues of high-resolution IMS-MS for the analysis of standard, as well as natural, ribonucleotide mixtures containing isomeric/isobaric species. Four methyladenosine monophosphates, four methylcytidine monophosphates, as well as various cyclic ribonucleotide isomers, were infused either separately or as mixtures into a prototype Q-TOF with cyclic IMS separator, and a Synapt G2 HDMS for comparison purposes. Preliminary experiments have shown that the cyclic IMS separator was capable of separating modified ribonucleotides by using one to three cycles (effective path length of 3 m). The mixture of four methylcytidines was first separated into three peaks after 9 spins. Then, the center pair (m3C and Cm) was fully separated using a Heartcut experiment, in which the pair was allowed to spin for another 270 ms after ejecting the leading and trailing peaks to prevent their overlap. All four methyladenosines, 2’,3’- and 3’,5’-cGMPs, and 2’,3’- and 3’,5’-cAMPs were separated by using the same approach but different numbers of spins and effective paths. We also tested the ability to perform tandem MS of mobility-resolved species to secure further PTM identification and quantification. Next, we plan on analyzing ribonucleotide mixtures obtained by exonuclease digestion of total RNA extracts to test the performance of this technique with actual samples of cellular origin.
Novel Aspect
Increasing path length of mobility separation resolves nucleotide isomers based on collisional cross section and fragmentation of mobility-resolved analytes Options:
Submitting Author:
Reza Nemati Josheghani University at Albany Albany, NY reza.nematy@gmail.com