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

2020; Nature Portfolio; Volume: 11; Issue: 1 Linguagem: Inglês

10.1038/s41467-020-19928-x

2041-1723

Danye Qiu, Miranda Wilson, Verena B. Eisenbeis, Robert K. Harmel, Esther Riemer, Thomas Haas, Christopher Wittwer, Nikolaus Jork, Chunfang Gu, Stephen B. Shears, Gabriel Schaaf, Bernd Kammerer, Dorothea Fiedler, Adolfo Saiardi, Henning J. Jessen,

Trace Elements in Health

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 [ 13 C 6 ]- myo -inositol or [ 13 C 6 ]- 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.