Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 22, Issue 8, Pages 1363-1372Publisher
SPRINGER
DOI: 10.1007/s13361-011-0137-5
Keywords
Pseudouridine quantification; tRNA; Mass spectrometry; Selected reaction monitoring; SRM; Detection of RNA modifications; Oligonucleotides
Funding
- National Institutes of Health [GM 58843]
- University of Cincinnati
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [0910751] Funding Source: National Science Foundation
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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.
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