An Analytical Platform for Mass Spectrometry-based Identification and Chemical Analysis of RNA in Ribonucleoprotein Complexes

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2009 Sep 11

PMID 19740761

Citations 22

Authors

Masato Taoka

Yoshio Yamauchi

Yuko Nobe

Shunpei Masaki

Hiroshi Nakayama

Hideaki Ishikawa

Nobuhiro Takahashi

Toshiaki Isobe

Affiliations

Soon will be listed here.

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.

Citing Articles

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Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry.

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Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling.

Bailey A, Talkish J, Ding H, Igel H, Duran A, Mantripragada S Elife. 2022; 11.

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Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

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