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

Taoka, Masato, Yamauchi, Yoshio, Nobe, Yuko, Masaki, Shunpei, Nakayama, Hiroshi, Ishikawa, Hideaki, Takahashi, Nobuhiro, Isobe, Toshiaki
Nucleic acids research 2009 v.37 no.21 pp. e140
chemical analysis, cultured cells, databases, dissociation, mass spectrometry, reversed-phase liquid chromatography, ribonucleoproteins, small interfering RNA, solvents, spectrometers, transfer RNA, yeasts
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.