title: Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry.
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.
authors.affiliation: 1] Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2].
authors.forename: Dan
authors.initials: D
authors.lastname: Su
authors.affiliation: 1] Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2].
authors.forename: Clement T Y
authors.initials: CT
authors.lastname: Chan
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Gu
authors.affiliation: 1] Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2].
authors.forename: Kok Seong
authors.initials: KS
authors.lastname: Lim
authors.affiliation: Singapore-MIT Alliance for Research and Technology, Campus for Research Excellence and Technical Enterprise (CREATE), Singapore.
authors.forename: Yok Hian
authors.initials: YH
authors.lastname: Chionh
authors.affiliation: Singapore-MIT Alliance for Research and Technology, Campus for Research Excellence and Technical Enterprise (CREATE), Singapore.
authors.forename: Megan E
authors.initials: ME
authors.lastname: McBee
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Russell
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors.forename: I Ramesh
authors.initials: IR
authors.lastname: Babu
authors.affiliation: College of Nanoscale Science and Engineering, University at Albany, State University of New York, Albany, New York, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Begley
authors.affiliation: 1] Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2] Singapore-MIT Alliance for Research and Technology, Campus for Research Excellence and Technical Enterprise (CREATE), Singapore. [3] Center for Environmental Health Science, MIT, Cambridge, Massachusetts, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D012343:RNA, Transfer / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D012263:Ribonucleosides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D012263:Ribonucleosides; D012343:RNA, Transfer