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dc.contributor.authorSu, Dan
dc.contributor.authorGu, Chen
dc.contributor.authorLim, Kok Seong
dc.contributor.authorChionh, Yok Hian
dc.contributor.authorChan, Clement T. Y.
dc.contributor.authorMcBee, Megan E.
dc.contributor.authorRussell, Brandon S.
dc.contributor.authorRamesh Babu, I.
dc.contributor.authorBegley, Thomas J.
dc.contributor.authorDedon, Peter C.
dc.date.accessioned2015-10-15T14:01:07Z
dc.date.available2015-10-15T14:01:07Z
dc.date.issued2014-03
dc.identifier.issn1754-2189
dc.identifier.issn1750-2799
dc.identifier.urihttp://hdl.handle.net/1721.1/99341
dc.description.abstractPost-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography–mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.en_US
dc.description.sponsorshipNational Institute of Environmental Health Sciences (ES002109)en_US
dc.description.sponsorshipNational Institute of Environmental Health Sciences (ES015037)en_US
dc.description.sponsorshipNational Institute of Environmental Health Sciences (ES017010)en_US
dc.description.sponsorshipWestaway Research Funden_US
dc.description.sponsorshipMassachusetts Institute of Technology (Merck Fellowship)en_US
dc.description.sponsorshipDavid H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship)en_US
dc.description.sponsorshipHoward Hughes Medical Institute (International Student Research Fellowship)en_US
dc.description.sponsorshipSingapore-MIT Alliance for Research and Technologyen_US
dc.language.isoen_US
dc.publisherNature Publishing Groupen_US
dc.relation.isversionofhttp://dx.doi.org/10.1038/nprot.2014.047en_US
dc.rightsArticle is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.en_US
dc.sourcePMCen_US
dc.titleQuantitative analysis of tRNA modifications by HPLC-coupled mass spectrometryen_US
dc.title.alternativeQuantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometryen_US
dc.typeArticleen_US
dc.identifier.citationSu, Dan, Clement T Y Chan, Chen Gu, Kok Seong Lim, Yok Hian Chionh, Megan E McBee, Brandon S Russell, I Ramesh Babu, Thomas J Begley, and Peter C Dedon. “Quantitative Analysis of Ribonucleoside Modifications in tRNA by HPLC-Coupled Mass Spectrometry.” Nature Protocols 9, no. 4 (March 13, 2014): 828–841.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Center for Environmental Health Sciencesen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biological Engineeringen_US
dc.contributor.mitauthorSu, Danen_US
dc.contributor.mitauthorChan, Clement T. Y.en_US
dc.contributor.mitauthorGu, Chenen_US
dc.contributor.mitauthorLim, Kok Seongen_US
dc.contributor.mitauthorRussell, Brandon S.en_US
dc.contributor.mitauthorRamesh Babu, I.en_US
dc.contributor.mitauthorDedon, Peter C.en_US
dc.relation.journalNature Protocolsen_US
dc.eprint.versionAuthor's final manuscripten_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dspace.orderedauthorsSu, Dan; Chan, Clement T Y; Gu, Chen; Lim, Kok Seong; Chionh, Yok Hian; McBee, Megan E; Russell, Brandon S; Babu, I Ramesh; Begley, Thomas J; Dedon, Peter Cen_US
dc.identifier.orcidhttps://orcid.org/0000-0003-0011-3067
dc.identifier.orcidhttps://orcid.org/0000-0001-8533-2706
dc.identifier.orcidhttps://orcid.org/0000-0001-7940-3459
dc.identifier.orcidhttps://orcid.org/0000-0001-9920-2080
mit.licensePUBLISHER_POLICYen_US


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