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An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

Author:
Taoka, Masato, Yamauchi, Yoshio, Nobe, Yuko, Masaki, Shunpei, Nakayama, Hiroshi, Ishikawa, Hideaki, Takahashi, Nobuhiro, Isobe, Toshiaki
Source:
Nucleic acids research 2009 v.37 no.21 pp. e140
ISSN:
0305-1048
Subject:
chemical analysis, cultured cells, databases, dissociation, mass spectrometry, reversed-phase liquid chromatography, ribonucleoproteins, small interfering RNA, solvents, spectrometers, transfer RNA, yeasts
Abstract:
We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ~21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.
Agid:
2213512