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With the ability to differentiate different molecular sizes, ion mobility spectrometry (IMS) has great potentials in the analysis of isomeric compounds. However, due to the lack of sensitivity and resolution, IMS has not been commonly used. To address the issue on resolution, the goals of this study are to explore a more effective way to perform IMS by reducing the size of ions prior to the IM measurements, and apply the new approach to the differentiation of isomeric RNA modifications. The size reduction of ribonucleoside ions was effectively accomplished by using the collision-induced dissociation process, in which the N-glycosidic bond in ribonucleoside was cleaved and split the ions into two parts—a smaller nucleobase ion and a neutral molecule of ribose sugar. Since the chemical group that corresponds to most of the RNA modifications makes up a relatively small part of the molecular structure of nucleobases, the differentiation of the dissociated nucleobase ions is expected to require a lower ion mobility resolution than the differentiation of bigger isomeric ribonucleoside ions. By using RNA methylation as a model in this study, the proposed method lowered the required resolution by 16% for the differentiation of 1-methyladenosine and N6-methyladenosine. Similar results were also obtained from the differentiation of methylated cytidine isomers. In comparison to the results obtained from using the conventional tandem mass spectrometric method, there was no significant loss of signals when the proposed method was used. The proposed method is expected to be applicable to other types of isomeric compounds. Also, the same approach is applicable on other IMS platforms.

Enhanced differentiation of isomeric RNA modifications by reducing the size of ions in ion mobility mass spectrometric measurements