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Nucleic Acids Res. 2009 Nov;37(21):e140. doi: 10.1093/nar/gkp732.

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

Author information

  • 1Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.

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 approximately 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.

PMID:
19740761
[PubMed - indexed for MEDLINE]
PMCID:
PMC2790879
Free PMC Article

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