title: Sequence mapping of transfer RNA chemical modifications by liquid chromatography tandem mass spectrometry.
abstract: Mass spectrometry is a powerful analytical tool for identifying and characterizing structural modifications to the four canonical bases in RNA, information that is lost when using techniques such as PCR for RNA analysis. Here we described an updated method for sequence mapping of modified nucleosides in transfer RNA. This modification mapping approach utilizes knowledge of the modified nucleosides present in the sample along with the genome-derived tRNA sequence to readily locate modifications site-specifically in the tRNA sequence. The experimental approach involves isolation of the tRNA of interest followed by separate enzymatic digestion to nucleosides and oligonucleotides. Both samples are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and the data sets are then combined to yield the modification profile of the tRNA. Data analysis is facilitated by the use of unmodified sequence exclusion lists and new developments in software that can automate MS/MS spectral annotation. The method is illustrated using tRNA-Asn isolated from Thermus thermophilus.
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Ross
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Cao
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States.
authors.forename: Ningxi
authors.initials: N
authors.lastname: Yu
authors.affiliation: Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States. Electronic address: Pat.Limbach@uc.edu.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics / Q000302:isolation & purification*; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*; D016963:Thermus thermophilus / Q000235:genetics
chemical_list: D012343:RNA, Transfer
keywords: LC–MS/MS; Modified bases; Modified nucleosides; RNA sequencing; Tandem mass spectrometry; tRNA