Improved phosphometabolome profiling applying isotope dilution strategy and capillary ion chromatography-tandem mass spectrometry

摘要

The phosphometabolome is comprised of all phosphorylated metabolites including the major metabolite classes sugar phosphates and nucleoside phosphates. Phosphometabolites are invaluable in any cell as a part of primary and energy-metabolism, and as building blocks in the biosynthesis of macromolecules. Here, we report quantitative profiling of the phosphometabolome by applying capillary ion chromatography-tandem mass spectrometry (caplC-MS/MS), ensuring improved chromatographic separation, robustness and quantitative precision. Baseline separation was achieved for six out of eight tested hexose phosphates. Quantitative precision and reproducibility was improved by introducing a fully uniformly (U) C-13-labeled biological extract and applying an isotope dilution (ID) correction strategy. A C-13-labeled biological extract does in principle contain internal standards (IS) for all metabolites, but low abundant metabolites pose a challenge, and solutions to this are discussed. The extreme reproducibility and reliability of this caplC-MS/MS method was demonstrated by running the instrumentation continuously for ten days.