title: Stable Isotope Labeling for Improved Comparative Analysis of RNA Digests by Mass Spectrometry.
abstract: Even with the advent of high throughput methods to detect modified ribonucleic acids (RNAs), mass spectrometry remains a reliable method to detect, characterize, and place post-transcriptional modifications within an RNA sequence. Here we have developed a stable isotope labeling comparative analysis of RNA digests (SIL-CARD) approach, which improves upon the original18O/16O labeling CARD method. Like the original, SIL-CARD allows sequence or modification information from a previously uncharacterized in vivo RNA sample to be obtained by direct comparison with a reference RNA, the sequence of which is known. This reference is in vitro transcribed using a13C/15N isotopically enriched nucleoside triphosphate (NTP). The two RNAs are digested with an endonuclease, the specificity of which matches the labeled NTP used for transcription. As proof of concept, several transfer RNAs (tRNAs) were characterized by SIL-CARD, where labeled guanosine triphosphate was used for the reference in vitro transcription. RNase T1 digestion products from the in vitro transcript will be 15 Da higher in mass than the same digestion products from the in vivo tRNA that are unmodified, leading to a doublet in the mass spectrum. Singlets, rather than doublets, arise if a sequence variation or a post-transcriptional modification is present that results in a relative mass shift different from 15 Da. Moreover, the use of the in vitro synthesized tRNA transcript allows for quantitative measurement of RNA abundance. Overall, SIL-CARD simplifies data analysis and enhances quantitative RNA modification mapping by mass spectrometry. Graphical Abstract ᅟ.
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, USA.
authors.forename: Mellie June
authors.initials: MJ
authors.lastname: Paulines
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: D002138:Calibration; D002247:Carbon Isotopes / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods* / Q000592:standards; D009587:Nitrogen Isotopes / Q000737:chemistry; D012313:RNA / Q000032:analysis* / Q000737:chemistry*; D012343:RNA, Transfer / Q000032:analysis / Q000737:chemistry; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012313:RNA; D012343:RNA, Transfer; D006163:Ribonuclease T1
keywords: LC-MS/MS; Modified nucleosides; RNA sequencing; Stable isotope labeling; Tandem mass spectrometry; tRNA