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Nucleic Acids Research Advance Access published online on September 9, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp732
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Masato Taoka1, Yoshio Yamauchi1, Yuko Nobe2, Shunpei Masaki2, Hiroshi Nakayama2,3, Hideaki Ishikawa2,4, Nobuhiro Takahashi2,4 and Toshiaki Isobe1,2,*

1Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, 2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, 3Biomolecular Characterization Team, RIKEN Advanced Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198 and 4Department of Biotechnology, United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu-shi, Tokyo 183-8509, Japan

*To whom correspondence should be addressed. Tel: +81 42 677 2542; Fax: +81 42 677 2525; Email: isobe-toshiaki{at}tmu.ac.jp

Received May 18, 2009. Revised August 18, 2009. Accepted August 19, 2009.

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.

The authors wish it to know that, in their opinion, the first two authors should be regarded as joint First Authors.


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