title: Applications and Advantages of Stable Isotope Phosphate Labeling of RNA in Mass Spectrometry.
abstract: Mass spectrometry (MS) has become an enabling technology for the characterization of post-transcriptionally modified nucleosides within ribonucleic acids (RNAs). These modified RNAs tend to be more challenging to completely characterize using conventional genomic-based sequencing technologies. As with many biological molecules, information relating to the presence or absence of a particular compound (i.e., qualitative measurement) is only one step in sample characterization. Additional useful information is found by performing quantitative measurements on the levels of the compound of interest in the sample. Phosphate labeling of modified RNAs has been developed by our laboratory to enhance conventional mass spectrometry techniques. By taking advantage of the mechanism of action of many ribonucleases (RNases), digesting RNA samples in the presence of18O-labeled water generates an18O-labeled 3'-phosphate in each digestion product. We describe the historical development of this approach, contrast this stable isotope labeling strategy with others used in RNA mass spectrometry, and provide examples of new analytical mass spectrometry methods that are enabled by phosphate labeling in this fashion.
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, USA.
authors.forename: Kayla
authors.initials: K
authors.lastname: Borland
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, USA. Pat.Limbach@uc.edu.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D007553:Isotope Labeling; D013058:Mass Spectrometry; D010710:Phosphates / Q000737:chemistry*; D012313:RNA / Q000032:analysis*
chemical_list: D010710:Phosphates; D012313:RNA
keywords: 18O-enriched water; LC–MS/MS; Modified nucleosides; RNA sequencing; Stable isotope labeling; tRNA