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Normalized retention time for scheduled liquid chromatography-multistage mass spectrometry analysis of epitranscriptomic modifications.
Metadata
Journaljournal of chromatography a4.049Date
2020 May 08
5 months ago
Type
Journal Article
Volume
2020-Jul-19 / 1623 : 461181
Author
Gonzalez G 1, Cui Y 1, Wang P 1, Wang Y 2
Affiliation
  • 2. Environmental Toxicology Graduate Program, United States; Department of Chemistry, University of California, Riverside, California 92521, United States. Electronic address: [email protected]
Doi
PMIDMESH
Chromatography, Liquid
Epigenesis, Genetic
Graphite
HEK293 Cells
Humans
Mass Spectrometry
Ribonucleosides
Time Factors
Transcriptome
Abstract
Investigations into post-transcriptional modifications of RNA and their regulatory proteins have revealed pivotal roles of these modifications in cellular functions. A robust method for the quantitative analysis of modified nucleosides in RNA may facilitate the assessment about their functions in RNA biology and disease etiology. Here, we developed a sensitive nano-liquid chromatography-multistage mass spectrometry (nLC-MS3) method for profiling simultaneously 27 modified ribonucleosides. We employed normalized retention time (iRT) and scheduled selected-reaction monitoring (SRM) to achieve high-throughput analysis, where we assigned iRT values for modified ribonucleosides based on their relative elution times with respect to the four canonical ribonucleosides. The iRT scores allowed for reliable predictions of retention times for modified ribonucleosides with the use of two types of stationary phase materials and various mobile phase gradients. The method enabled the identification of 20 modified ribonucleosides with the use of the enzymatic digestion mixture of 2.5 ng total RNA and facilitated robust quantification of modified cytidine derivatives in total RNA. Together, we established a scheduled SRM-based method for high-throughput analysis of modified ribonucleosides with the use of a few nanograms of RNA.
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J Chromatogr Ajournal of chromatography a
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