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A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

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    Research Article

    A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase
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    • Anders Michael Bernth Giessing
      Anders Michael Bernth Giessing
      Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark
      More by Anders Michael Bernth Giessing
    • Lincoln Greyson Scott
      Lincoln Greyson Scott
      Cassia LLC, 3030 Bunker Hill Street, Suite 214, 92109, San Diego, CA, USA
      More by Lincoln Greyson Scott
    • Finn Kirpekar
      Finn Kirpekar
      Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark
      More by Finn Kirpekar
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    Journal of The American Society for Mass Spectrometry

    Cite this: J. Am. Soc. Mass Spectrom. 2011, 22, 7, XXX-XXX
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    https://doi.org/10.1007/s13361-011-0126-8
    Published April 15, 2011
    Copyright © 2011 © American Society for Mass Spectrometry 2011
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    Abstract

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    LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nano-chip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ × 150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MSn identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh5C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh5C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh5C was readily identified by its MH+m/z 260 and MSn fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.

    Copyright © 2011 © American Society for Mass Spectrometry 2011

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    This article is cited by 14 publications.

    1. Guadalupe Espadas, Julia Morales-Sanfrutos, Rebeca Medina, Morghan C Lucas, Eva Maria Novoa, Eduard Sabidó. High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry. Journal of Chromatography A 2022, 1665 , 462803. https://doi.org/10.1016/j.chroma.2022.462803
    2. Pavlina Gregorova, Nina H. Sipari, L. Peter Sarin. Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS. RNA Biology 2021, 18 (10) , 1382-1389. https://doi.org/10.1080/15476286.2020.1853385
    3. Gwendolyn Gonzalez, Yuxiang Cui, Pengcheng Wang, Yinsheng Wang. Normalized retention time for scheduled liquid chromatography-multistage mass spectrometry analysis of epitranscriptomic modifications. Journal of Chromatography A 2020, 1623 , 461181. https://doi.org/10.1016/j.chroma.2020.461181
    4. Stephanie Stransky, Jennifer Aguilan, Jake Lachowicz, Carlos Madrid-Aliste, Edward Nieves, Simone Sidoli. Mass Spectrometry to Study Chromatin Compaction. Biology 2020, 9 (6) , 140. https://doi.org/10.3390/biology9060140
    5. Farideh Haghighi, Zahra Talebpour, Amir Sanati Nezhad. Towards fully integrated liquid chromatography on a chip: Evolution and evaluation. TrAC Trends in Analytical Chemistry 2018, 105 , 302-337. https://doi.org/10.1016/j.trac.2018.05.002
    6. Ed Dudley, Liz Bond. Mass spectrometry analysis of nucleosides and nucleotides. Mass Spectrometry Reviews 2014, 33 (4) , 302-331. https://doi.org/10.1002/mas.21388
    7. Magnus Rogeberg, Helle Malerod, Hanne Roberg-Larsen, Cecilie Aass, Steven Ray Wilson. On-line solid phase extraction–liquid chromatography, with emphasis on modern bioanalysis and miniaturized systems. Journal of Pharmaceutical and Biomedical Analysis 2014, 87 , 120-129. https://doi.org/10.1016/j.jpba.2013.05.006
    8. Lena Wicke, Joachim W. Engels. An unexpected methyl group migration during on-column Stille derivatization of RNA. Tetrahedron 2014, 70 (2) , 327-333. https://doi.org/10.1016/j.tet.2013.11.061
    9. Zhiwei Qin, Alexander Thomas Baker, Andrea Raab, Sheng Huang, Tiehui Wang, Yi Yu, Marcel Jaspars, Christopher J. Secombes, Hai Deng. The Fish Pathogen Yersinia ruckeri Produces Holomycin and Uses an RNA Methyltransferase for Self-resistance. Journal of Biological Chemistry 2013, 288 (21) , 14688-14697. https://doi.org/10.1074/jbc.M112.448415
    10. Reiko Sakaguchi, Anders Giessing, Qing Dai, Georges Lahoud, Zita Liutkeviciute, Saulius Klimasauskas, Joseph Piccirilli, Finn Kirpekar, Ya-Ming Hou. Recognition of guanosine by dissimilar tRNA methyltransferases. RNA 2012, 18 (9) , 1687-1701. https://doi.org/10.1261/rna.032029.111
    11. Line H.G. Larsen, Anette Rasmussen, Anders M.B. Giessing, Gerwald Jogl, Finn Kirpekar. Identification and Characterization of the Thermus thermophilus 5-Methylcytidine (m5C) Methyltransferase Modifying 23 S Ribosomal RNA (rRNA) Base C1942. Journal of Biological Chemistry 2012, 287 (33) , 27593-27600. https://doi.org/10.1074/jbc.M112.376160
    12. Anders M.B. Giessing, Finn Kirpekar. Mass spectrometry in the biology of RNA and its modifications. Journal of Proteomics 2012, 75 (12) , 3434-3449. https://doi.org/10.1016/j.jprot.2012.01.032
    13. Klaus B. Mogensen, Jörg P. Kutter. Carbon nanotube based stationary phases for microchip chromatography. Lab on a Chip 2012, 12 (11) , 1951. https://doi.org/10.1039/c2lc40102a
    14. Jesper Foged Havelund, Anders Michael Bernth Giessing, Trine Hansen, Anette Rasmussen, Lincoln Greyson Scott, Finn Kirpekar. Identification of 5-Hydroxycytidine at Position 2501 Concludes Characterization of Modified Nucleotides in E. coli 23S rRNA. Journal of Molecular Biology 2011, 411 (3) , 529-536. https://doi.org/10.1016/j.jmb.2011.06.036
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    Journal of The American Society for Mass Spectrometry

    Cite this: J. Am. Soc. Mass Spectrom. 2011, 22, 7, XXX-XXX
    Click to copy citationCitation copied!
    https://doi.org/10.1007/s13361-011-0126-8
    Published April 15, 2011
    Copyright © 2011 © American Society for Mass Spectrometry 2011
    Request reuse permissions

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