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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 197; p. 113943
• Main Authors: Kogaki, Takahiro, Ohshio, Ikumi, Ura, Hasumi, Iyama, Souta, Kitae, Kaori, Morie, Toshiya, Fujii, Shintarou, Sato, Shohei, Nagata, Toshiyuki, Takeda, Aya Harada, Aoki, Masaya, Ueda, Kazuhiro, Minami, Kentaro, Yamamoto, Masatatsu, Kawahara, Kohichi, Furukawa, Tatsuhiko, Sato, Masami, Ueda, Yuko, Jingushi, Kentaro, Tozuka, Zenzaburo, Saigusa, Daisuke, Hase, Hiroaki, Tsujikawa, Kazutake
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-04-2021
• Subjects: Modified nucleosides; Non-small cell lung cancer; Ribonucleic acid; UHPLC-MS/MS; UniSpray; UniSpray; UHPLC-MS/MS; Modified nucleosides; Ribonucleic acid; Non-small cell lung cancer
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.113943

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•We developed a novel method to detect modified nucleosides by combining a new ion source (UniSpray) with UHPLC-MS/MS.•UniSpray could detect nucleosides with higher sensitivity than ESI.•This method was capable of detecting 46 kinds of modified nucleosides simultaneously in 15 min.•The LOQ of this method was between 10 fg and 5 pg for 46 different nucleosides.•Nucleosides in RNA extracted from the mouse tissues and NSCLC specimens were measured by developed method.
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.