Mass Spectrometry-Based Quantification of Pseudouridine in RNA

作者：Addepalli Balasubrahmanyam; Limbach Patrick A^*

来源：Journal of the American Society for Mass Spectrometry, 2011, 22(8): 1363-1372.

DOI：10.1007/s13361-011-0137-5

• Pseudouridine quantification
• tRNA
• Mass spectrometry
• Selected reaction monitoring
• SRM
• Detection of RNA modifications
• Oligonucleotides

Direct detection of pseudouridine (psi), an isomer of uridine, in RNA is challenging. The most popular method requires chemical derivatization using N-cyclohexyl-N'-beta-(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.

• 出版日期2011-8