Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs

• Published in: Journal of bioscience and bioengineering Vol. 113; no. 3; pp. 412 - 419
• Main Authors: Izumi, Yoshihiro, Takimura, Shin, Yamaguchi, Shinichi, Iida, Junko, Bamba, Takeshi, Fukusaki, Eiichiro
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2012; Elsevier
• Subjects: Biological and medical sciences; Biotechnology; Chemically-synthesized small RNA; Data processing; dissociation; Electrospray ionization ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS); Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; ionization; ions; mass spectrometry; MicroRNA; MicroRNAs - analysis; MicroRNAs - chemistry; Multistep MSn analysis; RNA; RNA - analysis; RNA - chemistry; RNA modification; Sensitivity and Specificity; Sequence Analysis; Sequence Analysis, RNA - methods; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; RNA modification; MicroRNA; Multistep MSn analysis; Chemically-synthesized small RNA; Electrospray ionization ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS); Gene silencing; RNA; Micro RNA; analysis; Application; Mass spectrometry; Electrospray; Multistep MS
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2011.11.007

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-IT/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 (MS1). The ion exhibited high mass accuracy (<7ppm) and high mass resolution (a value of m/Δm=10,000) and was therefore very useful in RNA composition assignment. The optimized MS2 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 MS3 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 3min and 100nM, 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.