Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs(METHODS)
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概要
- 論文の詳細を見る
In this study, we have demonstrated an accurate and rapid small RNA analytical method with both sequence determination and detailed modification analysis by electrospray ionization-ion trap/time-of-flight mass spectrometry (ESI-lT/TOFMS). To develop this ideal method, we have examined the performance of ESI-IT/TOFMS using various chemically-synthesized model sequences of modified or unmodified microRNAs (miRNAs). The deconvoluted mass of a 22-nucleotide (nt) miRNA was obtained from a multiply charged precursor ion (MS^1). The ion exhibited high mass accuracy (<7 ppm) and high mass resolution (a value of m/Δm = 10,000) and was therefore very useful in RNA composition assignment. The optimized MS^2 method using ion trap collision-induced dissociation, as well as automatic annotation analysis of product ions based on the accurate mass information, enabled the precise sequencing determination of intact miRNAs. Further, the detailed structural analysis of 3'-terminal modified nucleic acid in intact methylated miRNA was carried out using the MS^3 capability of the hybrid IT/TOFMS. The direct infusion method also provided a high throughput and good sensitivity because the analytical time and sample concentration needed in a series of experiments with reliable data were only 3 min and 100 nM, respectively. This study provides a novel approach for characterizing the intact chemically-synthesized small RNA without chemical and enzymatic digestions and would be widely applicable for the structural analysis of complicated modified small RNAs.
著者
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FUKUSAKI EI-ICHIRO
Department of Biotechnology, Graduate School of Engineering, Osaka University
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Bamba Takeshi
Department Of Applied Environmental Biology Graduate School Of Pharmaceutical Sciences Osaka Univers
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Bamba Takeshi
Dep. Of Biotechnology Graduate School Of Engineering Osaka Univ. 2- 1 Yamadaoka Suita Osaka 565-0871
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Bamba Takeshi
Dept. Biotech. Grad. Sch. Eng. Osaka Univ.:hitachi Zosen Co.
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IIDA Junko
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University
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Fukusaki Eiichiro
Division Of Advanced Science And Biotechnology Graduate School Of Engineering Osaka University
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Fukusaki Eiichiro
Dep. Of Biotechnology Graduate School Of Engineering Osaka Univ.
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Fukusaki Ei-ichiro
大阪大学大学院
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Fukusaki Eiichiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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Fukusaki Eiichiro
Dept. Mat. Life Sci. Osaka Univ
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Izumi Yoshihiro
Dept. Biotech. Grad. Sch. Eng. Osaka Univ.
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Izumi Yoshihiro
Department Of Biological Science Faculty Of Science Kumamoto University
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Yamaguchi Shinichi
Department Of Applied Chemistry Faculty Of Science & Technology Kinki University
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Takimura Shin
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Iida Junko
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Yamaguchi Shinichi
Department of Biotechnology, Graduate School of Engineering, Osaka University
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