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Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism

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Abstract

Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.

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Acknowledgments

We acknowledge Ralf Falter (Agilent Technologies, Waldbronn, Germany) for the support and fruitful discussions. We would like to thank Tilman Brummer (Albert-Ludwigs-University Freiburg, Germany) for providing the breast epithelial cell line MCF-10A and the technical support.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, Habsburgerstr. 49, 79104, Freiburg, Germany

    Lucas Willmann & Bernd Kammerer

  2. Agilent Technologies, Hewlett-Packard-Straße 8, 76337, Waldbronn, Germany

    Sonja Krieger, Jens Trafkowski & Michael Rodamer

  3. Institute of Biology II, Albert-Ludwigs-University Freiburg, Schänzlestr. 1, 79104, Freiburg, Germany

    Lucas Willmann

  4. University Medical Center Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany

    Thalia Erbes

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  1. Lucas Willmann
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Correspondence to Bernd Kammerer.

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Willmann, L., Erbes, T., Krieger, S. et al. Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism. Anal Bioanal Chem 407, 3555–3566 (2015). https://doi.org/10.1007/s00216-015-8516-6

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  • DOI: https://doi.org/10.1007/s00216-015-8516-6

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