An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes.
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.
Taoka
Masato
M
Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.
Yamauchi
Yoshio
Y
Nobe
Yuko
Y
Masaki
Shunpei
S
Nakayama
Hiroshi
H
Ishikawa
Hideaki
H
Takahashi
Nobuhiro
N
Isobe
Toshiaki
T
D056148:Chromatography, Reverse-Phase / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D009843:Oligoribonucleotides / Q000737:chemistry; D012331:RNA, Fungal / Q000737:chemistry; D034741:RNA, Small Interfering / Q000737:chemistry; D012342:RNA, Small Nuclear / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry; D022661:RNA, Untranslated / Q000737:chemistry*; D006163:Ribonuclease T1; D012261:Ribonucleoproteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D009843:Oligoribonucleotides; D012331:RNA, Fungal; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D012360:RNA, Transfer, Phe; D022661:RNA, Untranslated; D012261:Ribonucleoproteins; D006163:Ribonuclease T1