dc.contributor.author | Su, Dan | |
dc.contributor.author | Gu, Chen | |
dc.contributor.author | Lim, Kok Seong | |
dc.contributor.author | Chionh, Yok Hian | |
dc.contributor.author | Chan, Clement T. Y. | |
dc.contributor.author | McBee, Megan E. | |
dc.contributor.author | Russell, Brandon S. | |
dc.contributor.author | Ramesh Babu, I. | |
dc.contributor.author | Begley, Thomas J. | |
dc.contributor.author | Dedon, Peter C. | |
dc.date.accessioned | 2015-10-15T14:01:07Z | |
dc.date.available | 2015-10-15T14:01:07Z | |
dc.date.issued | 2014-03 | |
dc.identifier.issn | 1754-2189 | |
dc.identifier.issn | 1750-2799 | |
dc.identifier.uri | http://hdl.handle.net/1721.1/99341 | |
dc.description.abstract | Post-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.sponsorship | National Institute of Environmental Health Sciences (ES002109) | en_US |
dc.description.sponsorship | National Institute of Environmental Health Sciences (ES015037) | en_US |
dc.description.sponsorship | National Institute of Environmental Health Sciences (ES017010) | en_US |
dc.description.sponsorship | Westaway Research Fund | en_US |
dc.description.sponsorship | Massachusetts Institute of Technology (Merck Fellowship) | en_US |
dc.description.sponsorship | David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship) | en_US |
dc.description.sponsorship | Howard Hughes Medical Institute (International Student Research Fellowship) | en_US |
dc.description.sponsorship | Singapore-MIT Alliance for Research and Technology | en_US |
dc.language.iso | en_US | |
dc.publisher | Nature Publishing Group | en_US |
dc.relation.isversionof | http://dx.doi.org/10.1038/nprot.2014.047 | en_US |
dc.rights | Article 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.source | PMC | en_US |
dc.title | Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry | en_US |
dc.title.alternative | Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry | en_US |
dc.type | Article | en_US |
dc.identifier.citation | Su, 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.department | Massachusetts Institute of Technology. Center for Environmental Health Sciences | en_US |
dc.contributor.department | Massachusetts Institute of Technology. Department of Biological Engineering | en_US |
dc.contributor.mitauthor | Su, Dan | en_US |
dc.contributor.mitauthor | Chan, Clement T. Y. | en_US |
dc.contributor.mitauthor | Gu, Chen | en_US |
dc.contributor.mitauthor | Lim, Kok Seong | en_US |
dc.contributor.mitauthor | Russell, Brandon S. | en_US |
dc.contributor.mitauthor | Ramesh Babu, I. | en_US |
dc.contributor.mitauthor | Dedon, Peter C. | en_US |
dc.relation.journal | Nature Protocols | en_US |
dc.eprint.version | Author's final manuscript | en_US |
dc.type.uri | http://purl.org/eprint/type/JournalArticle | en_US |
eprint.status | http://purl.org/eprint/status/PeerReviewed | en_US |
dspace.orderedauthors | Su, 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 C | en_US |
dc.identifier.orcid | https://orcid.org/0000-0003-0011-3067 | |
dc.identifier.orcid | https://orcid.org/0000-0001-8533-2706 | |
dc.identifier.orcid | https://orcid.org/0000-0001-7940-3459 | |
dc.identifier.orcid | https://orcid.org/0000-0001-9920-2080 | |
mit.license | PUBLISHER_POLICY | en_US |
mit.metadata.status | Complete | |