NDLI: Ribonucleoside labeling with Os(vi): A methodological approach to evaluation of RNA methylation by HPLC-ICP-MS

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Metallomics Vol -1

Ribonucleoside labeling with Os(vi): A methodological approach to evaluation of RNA methylation by HPLC-ICP-MS

Content Provider	Royal Society of Chemistry (RSC)
Author	Flores, Crescencio Rodríguez Chan, Qilin Wrobel, Katarzyna Wrobel, Kazimierz
Copyright Year	2010
Abstract	Covalent modifications of nucleobases are thought to play an important role in regulating the functions of DNA and various cellular RNA types. Perhaps the best characterized is DNA methylation on cytosine (methyl tag attached to carbon 5 position) and such modification has also been detected in stable and long-lived RNA molecules. In this work, we propose a novel procedure enabling very sensitive quantification of methylcytidine and other ribonucleosides, based on reversed phase liquid chromatography with inductively coupled plasma mass spectrometry (ICP-MS) detection. The procedure relies on labeling ribose residues with osmium, by formation of a ternary complex between cis-diol ribose groups, hexavalent osmium (K2OsO2(OH)4) and tetramethylethylenediamine (TEMED). The derivatization reaction was carried out with 50 : 1 molar excess of Os to ribonucleoside, pH 4, for 2 h at room temperature. The structures of Os-labeled cytidine and methylcytidine were confirmed by electrospray ionization mass spectrometry. The separation of Os-labeled cytidine (C), uridine (U), 5-methylcytidine (5mC) and guanosine (G) was achieved on C18 column (Gemini, 150 × 3 mm, 5 μm) with isocratic elution (0.05% triethylamine + 6 mmol L−1 ammonium acetate, pH 4.4: methanol (85 : 15)) and a total flow rate 0.6 mL min−1. The column effluent was on-line introduced to ICP-MS (a model 7500 ce, Agilent Technologies) for specific detection at 189Os. Calibration was performed within the concentration range 0–200 nmol L−1 of each ribonucleoside and the analytical figures of merit were evaluated. For 100 μL injection, the detection limits for C, U, 5mC, G were 24, 38, 21 and 28 pmol L−1, respectively. While introducing Os(VI)-TEMED to the column, it eluted in the dead volume and the detection limit for osmium was 20 pmol L−1. The results obtained in this work might be helpful in the analysis of RNA digests, providing quantitative data on the ribonucleoside composition and RNA methylation (measured as the percentage of methylated cytidines with respect to total RNA cytidines).
Starting Page	140
Ending Page	146
Page Count	7
File Format	HTM / HTML PDF
ISSN	17565901
Volume Number	2
Issue Number	2
Journal	Metallomics
DOI	10.1039/b915474d
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	RNA Guanosine 5-Methylcytidine Ribose Project Gemini Triethylamine DNA methylation Cytosine Tetramethylethylenediamine Methanol Electrospray ionization Valence (chemistry) Concentration Carbon Chromatography Osmium Cytidine Ammonia Ionization Agilent Technologies DNA Micrometre PH Uridine Inductively coupled plasma mass spectrometry Ribonucleoside Methylation Mass spectrometry
Content Type	Text
Resource Type	Article
Subject	Chemistry Medicine Metals and Alloys Biochemistry Biomaterials Biophysics

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Ribonucleoside labeling with Os(vi): A methodological approach to evaluation of RNA methylation by HPLC-ICP-MS