title: Mass spectrometry-based quantification of pseudouridine in RNA.
abstract: Direct detection of pseudouridine (ψ), an isomer of uridine, in RNA is challenging. The most popular method requires chemical derivatization using N-cyclohexyl-N'-β-(4-methylmorpholinum ethyl) carbodiimide p-tosylate (CMCT) followed by radiolabeled primer extension mediated by reverse transcriptase. More recently, mass spectrometry (MS)-based approaches for sequence placement of pseudouridine in RNA have been developed. Nearly all of these approaches, however, only yield qualitative information regarding the presence or absence of pseudouridine in a given RNA population. Here, we have extended a previously developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to enable both the qualitative and quantitative analysis of pseudouridine. Quantitative selected reaction monitoring (SRM) assays were developed using synthetic oligonucleotides, with or without pseudouridine, and the results yielded a linear relationship between the ion abundance of the pseudouridine-specific fragment ion and the amount of pseudouridine-containing oligonucleotide present in the original sample. Using this quantitative SRM assay, the extent of pseudouridine hypomodification in the conserved T-loop of tRNA isolated from two different Escherichia coli strains was established.
authors.affiliation: Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
authors.forename: Balasubrahmanyam
authors.initials: B
authors.lastname: Addepalli
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
mesh_terms: D001483:Base Sequence; D003028:CME-Carbodiimide / Q000031:analogs & derivatives; D002138:Calibration; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D016018:Least-Squares Analysis; D008969:Molecular Sequence Data; D011560:Pseudouridine / Q000032:analysis*; D012329:RNA, Bacterial / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D055672:Static Electricity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D012329:RNA, Bacterial; D011560:Pseudouridine; C060331:1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide; D003028:CME-Carbodiimide; D012343:RNA, Transfer