Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry

Danye Qiu (), Miranda S. Wilson, Verena B. Eisenbeis, Robert K. Harmel, Esther Riemer, Thomas M. Haas, Christopher Wittwer, Nikolaus Jork, Chunfang Gu, Stephen B. Shears, Gabriel Schaaf, Bernd Kammerer, Dorothea Fiedler, Adolfo Saiardi () and Henning J. Jessen ()
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Danye Qiu: Institute of Organic Chemistry, University of Freiburg
Miranda S. Wilson: Medical Research Council, Laboratory for Molecular Cell Biology, University College London
Verena B. Eisenbeis: Institute of Organic Chemistry, University of Freiburg
Robert K. Harmel: Leibniz-Forschungsinstitut für Molekulare Pharmakologie
Esther Riemer: Institute of Crop Science and Resource Conservation, Department of Plant Nutrition, Rheinische Friedrich-Wilhelms-University Bonn
Thomas M. Haas: Institute of Organic Chemistry, University of Freiburg
Christopher Wittwer: Institute of Organic Chemistry, University of Freiburg
Nikolaus Jork: Institute of Organic Chemistry, University of Freiburg
Chunfang Gu: Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health
Stephen B. Shears: Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health
Gabriel Schaaf: Institute of Crop Science and Resource Conservation, Department of Plant Nutrition, Rheinische Friedrich-Wilhelms-University Bonn
Bernd Kammerer: Institute of Organic Chemistry, University of Freiburg
Dorothea Fiedler: Leibniz-Forschungsinstitut für Molekulare Pharmakologie
Adolfo Saiardi: Medical Research Council, Laboratory for Molecular Cell Biology, University College London
Henning J. Jessen: Institute of Organic Chemistry, University of Freiburg

Nature Communications, 2020, vol. 11, issue 1, 1-12

Abstract: Abstract The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.

Date: 2020
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DOI: 10.1038/s41467-020-19928-x

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