Mass SpectrometryBased Quantification of Pseudour

Authors: Balasubrahmanyam Addepalli Patrick A Limbach

Publish Date: 2011/05/03

Volume: 22, Issue: 8, Pages: 1363-1372

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Abstract

Direct detection of pseudouridine ψ an isomer of uridine in RNA is challenging The most popular method requires chemical derivatization using NcyclohexylNβ4methylmorpholinum ethyl carbodiimide ptosylate CMCT followed by radiolabeled primer extension mediated by reverse transcriptase More recently mass spectrometry MSbased 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 LCMS/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 pseudouridinespecific fragment ion and the amount of pseudouridinecontaining oligonucleotide present in the original sample Using this quantitative SRM assay the extent of pseudouridine hypomodification in the conserved Tloop of tRNA isolated from two different Escherichia coli strains was establishedPseudouridine ψ is the most prevalent posttranscriptional modification in ribonucleic acids RNA 1 2 This isomer of uridine is commonly found in noncoding RNAs including transfer RNA tRNA ribosomal RNA rRNA small nuclear RNA snRNA and small nucleolar RNA snoRNA Moreover pseudouridine is found in all phylogenetic domains of life Archaea Eubacteria and Eukarya 3 Although a precise molecular role for this modification is unclear pseudouridine has been shown to confer rigidity and stabilize RNA structure enhance local base stacking and is involved in imino protonH2O coordination 4 5 Pseudouridine modulates codon–anticodon interactions between messenger RNA and tRNA 6 and facilitates ribosome assembly 7Pseudouridine is formed by sitespecific isomerization at the polyribonucleotide level following recognition of the target uridine in the context of the RNA sequence or structure of the site of interest by a group of enzymes called pseudouridine synthases 8 9 Studies on prokaryotes and eukaryotes have shown that deletion of pseudouridine synthase genes has a negative impact on cell growth and protein synthesis rates 10 11 indicating the critical function of this modification in the translational apparatus However identifying the exact location of pseudouridine in the context of its parent RNA is key to an improved understanding of the function of this posttranscriptional modificationPseudouridine is a masssilent modification therefore it does not exhibit a mass shift compared with its unmodified counterpart upon posttranscriptional formation in RNA The most widely used method for detection and sequence placement of pseudouridine is based on ψspecific irreversible derivatization using the reagent NcyclohexylNβ4methylmorpholinium ethylcarbodiimide ptosylate CMCT followed by reverse transcriptase mediated extension of radiolabeled primer and subsequent separation on a denaturing polyacrylamide gel 12 Although effective this method has some drawbacks and limitations which have spurred development of mass spectrometrybased approaches for the detection and sequence placement of pseudouridine in RNA 13 The two major mass spectrometrybased approaches involve enzymatic digestion of RNA to either nucleosides or oligonucleotides yielding samples suitable for analysisThe presence of pseudouridine can be detected by total enzymatic hydrolysis of RNA into nucleosides and subsequent analysis by liquid chromatography/electrospray ionization mass spectrometry LCESIMS 14 15 The sensitivity and specificity of pseudouridine detection in a RNA nucleoside mixture was further enhanced upon derivatization with methyl vinyl sulfone 15 A limitation of this approach is the lack of information relating to the sequence location of pseudouridine in the RNATo determine the specific sequence placement of this modification RNase mapping was employed in combination with chemical derivatization 16 17 18 RNase mapping uses basespecific endoribonucleases that generate a mixture of oligoribonucleotides which are amenable to accurate mass measurements by ESI or MALDI matrixassisted laser desorption/ionization mass spectrometry As pseudouridine is a mass silent modification the RNA before or after enzymatic treatment is chemically derivatized with either CMCT 16 17 or acrylonitrile cyanoethylation 18 to covalently link a mass tag to pseudouridine that enables differentiation of pseudouridine from uridine by mass spectrometry Identification of pseudouridine oligonucleotides is accomplished by mass spectrometric comparison of untreated and CMCT or acrylonitrile treated samples Mass differences of 252 Da CMCTtreated or 53 Da acrylonitrile between the two samples are used to identify RNase digestion products that contain pseudouridine 16 17 18A more recent development is a tandem mass spectrometry MS/MS approach compatible with LCMS based RNase mapping of nucleic acids 19 This approach was developed to take advantage of the unique but stable CC glycosidic bond which contrasts with the typically labile C–N glycosidic bond in uridine or other nucleosides The stable C–C glycosidic bond yields unique fragmentation pathways during collisioninduced dissociation CID leading to the production of pseudouridinespecific fragment ions These diagnostic ions include the doubly dehydrated nucleoside anion m/z 207 and its MS/MS product ion at m/z 164 as well as the fragmentation product at m/z 165 arising from MS/MS of m/z 225 Selective detection of these fragmentation pathways enabled the identification of pseudouridine in a given oligonucleotide by selected reaction monitoring SRM Specifically pseudouridines can be identified by scanning the SRM transition of m/z 207 to 164 Alternatively the SRM transition of m/z 225 to 165 can be used to selectively identify the presence of a 5 terminal pseudouridine in the RNase digestion product 19 McCloskey and coworkers used these SRM assays and chemical derivatization to identify pseudouridines in 23 S rRNAs of Haloarcula marismortui and Deinococcus radiodurans 20 and the 16 S rRNA of Thermus thermophilus 21

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