title: A novel method for sequence placement of modified nucleotides in mixtures of transfer RNA.
abstract: Sequence placement of post-transcriptionally modified nucleosides in tRNA can be experimentally difficult, particularly in cases involving new or unexpected modifications or sequence sites. We describe a mass spectrometry-based approach to this problem, involving the following steps: crude isolations of one or several tRNAs by HPLC from an unfractionated tRNA mixture; digestion to oligonucleotide mixtures by RNase T1; analysis by combined HPLC/electrospray ionization-MS for recognition of modifications; and direct gas-phase sequencing of selected targets in the mixture by LC/MS/MS. Isoacceptor identity can be established in favorable cases when tRNA gene sequences are available.
authors.affiliation: Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Wagner
authors.forename: Vinod
authors.initials: V
authors.lastname: Nair
authors.forename: Rebecca
authors.initials: R
authors.lastname: Guymon
authors.forename: Steven C
authors.initials: SC
authors.lastname: Pomerantz
authors.forename: Pamela F
authors.initials: PF
authors.lastname: Crain
authors.forename: Darrell R
authors.initials: DR
authors.lastname: Davis
authors.forename: James A
authors.initials: JA
authors.lastname: McCloskey
mesh_terms: D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D009711:Nucleotides / Q000032:analysis*; D012343:RNA, Transfer / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017423:Sequence Analysis, RNA / Q000379:methods*; D016944:Sulfolobus
chemical_list: D009711:Nucleotides; D012343:RNA, Transfer; D006163:Ribonuclease T1