Mass Spectrometry-Based Quantification of Pseudouridine in RNA

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 transcri...

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Veröffentlicht in:Journal of the American Society for Mass Spectrometry 2011-08, Vol.22 (8), p.1363-1372
Hauptverfasser: Addepalli, Balasubrahmanyam, Limbach, Patrick A.
Format: Artikel
Sprache:eng
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Zusammenfassung: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.
ISSN:1044-0305
1879-1123
DOI:10.1007/s13361-011-0137-5