Development of a highly sensitive method for the quantitative analysis of modified nucleosides using UHPLC-UniSpray-MS/MS.

Autor: Kogaki T; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Ohshio I; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Ura H; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Iyama S; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Kitae K; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Morie T; Center for Supporting Drug Discovery and Life Science Research Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Fujii S; Center for Supporting Drug Discovery and Life Science Research Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Sato S; Center for Supporting Drug Discovery and Life Science Research Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Nagata T; Department of General Thoracic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8520, Japan., Takeda AH; Department of General Thoracic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8520, Japan., Aoki M; Department of General Thoracic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8520, Japan., Ueda K; Department of General Thoracic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8520, Japan., Minami K; Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan., Yamamoto M; Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan., Kawahara K; Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan., Furukawa T; Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan., Sato M; Department of General Thoracic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8520, Japan., Ueda Y; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Jingushi K; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Tozuka Z; Center for Supporting Drug Discovery and Life Science Research Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan., Saigusa D; Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, 1-2 Seiryocho, Aobaku, Sendai, Miyagi 980-8573, Japan., Hase H; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: hase-h@phs.osaka-u.ac.jp., Tsujikawa K; Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2021 Apr 15; Vol. 197, pp. 113943. Date of Electronic Publication: 2021 Feb 02.
DOI: 10.1016/j.jpba.2021.113943
Abstrakt: There are more than 150 types of naturally occurring modified nucleosides, which are believed to be involved in various biological processes. Recently, an ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) technique has been developed to measure low levels of modified nucleosides. A comprehensive analysis of modified nucleosides will lead to a better understanding of intracellular ribonucleic acid modification, but this analysis requires high-sensitivity measurements. In this perspective, we established a highly sensitive and quantitative method using the newly developed ion source, UniSpray. A mass spectrometer was used with a UniSpray source in positive ion mode. Our UHPLC-UniSpray-MS/MS methodology separated and detected the four major nucleosides, 42 modified nucleosides, and dG15N5 (internal standard) in 15 min. The UniSpray method provided good correlation coefficients (>0.99) for all analyzed nucleosides, and a wide range of linearity for 35 of the 46 nucleosides. Additionally, the accuracy and precision values satisfied the criteria of <15% for higher concentrations and <20% for the lowest concentrations of all nucleosides. We also investigated whether this method could measure nucleosides in biological samples using mouse tissues and non-small cell lung cancer clinical specimens. We were able to detect 43 and 31 different modified nucleosides from mouse and clinical tissues, respectively. We also found significant differences in the levels of N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), 1-methylinosine (m1I), 2'-O-methylcytidine (Cm), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5S2U), and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) between cancerous and noncancerous tissues. In conclusion, we developed a highly sensitive methodology using UHPLC-UniSpray-MS/MS to simultaneously detect and quantify modified nucleosides, which can be used for analysis of biological samples.
Competing Interests: Declaration of Competing Interest The Authors declare that there is no conflict of interest.
(Copyright © 2021. Published by Elsevier B.V.)
Databáze: MEDLINE
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