Dataset Information

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.

SUBMITTER: Addepalli B

PROVIDER: S-EPMC3521532 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Mass spectrometry-based quantification of pseudouridine in RNA.

Addepalli Balasubrahmanyam B Limbach Patrick A PA

Journal of the American Society for Mass Spectrometry 20110503 8

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 ...[more]

PMID: 21953190

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Project description:ConspectusIn recent years, there has been a high interest in researching RNA modifications, as they are involved in many cellular processes and in human diseases. A substantial set of enzymes within the cell, called RNA writers, place RNA modifications selectively and site-specifically. Another set of enzymes, called readers, recognize these modifications which guide the fate of the modified RNA. Although RNA is a transient molecule and RNA modification could be removed by RNA degradation, a subclass of enzymes, called RNA erasers, remove RNA modifications selectively and site-specifically to alter the characteristics of the RNA. The detection of RNA modifications can be done by various methods including second and next generation sequencing but also mass spectrometry. An approach capable of both qualitative and quantitative RNA modification analysis is liquid chromatography coupled to mass spectrometry of enzymatic hydrolysates of RNA into nucleosides. However, for successful detection and quantification, various factors must be considered to avoid biased identification and inaccurate quantification. In this Account, we identify three classes of errors that may distort the analysis. These classes comprise (I) errors related to chemical instabilities, (II) errors revolving around enzymatic hydrolysis to nucleosides, and (III) errors arising from issues with chromatographic separation and/or subsequent mass spectrometric analysis.A prominent example for class 1 is Dimroth rearrangement of m1A to m6A, but class 1 also comprises hydrolytic reactions and reactions with buffer components. Here, we also present the conversion of m3C to m3U under mild alkaline conditions and propose a practical solution to overcome these instabilities. Class 2 errors-such as contaminations in hydrolysis reagents or nuclease specificities-have led to erroneous discoveries of nucleosides in the past and possess the potential for misquantification of nucleosides. Impurities in the samples may also lead to class 3 errors: For instance, issues with chromatographic separation may arise from residual organic solvents, and salt adducts may hamper mass spectrometric quantification. This Account aims to highlight various errors connected to mass spectrometry analysis of nucleosides and presents solutions for how to overcome or circumnavigate those issues. Therefore, the authors anticipate that many scientists, but especially those who plan on doing nucleoside mass spectrometry, will benefit from the collection of data presented in this Account as a raised awareness, toward the variety of potential pitfalls, may further enhance the quality of data.

Dataset Information

Mass spectrometry-based quantification of pseudouridine in RNA.

Publications

Mass spectrometry-based quantification of pseudouridine in RNA.

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