期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 174, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/62847
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资金
- European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [864246]
- Brigitte-Schlieben-Lange-Programm
- MRC program [MR/T028904/1]
- MRC [MR/T028904/1] Funding Source: UKRI
- European Research Council (ERC) [864246] Funding Source: European Research Council (ERC)
Inositol pyrophosphates are important intracellular signaling molecules, and a detailed protocol for their quantitation in mammalian cells using CE-ESI-MS has been presented. This method allows sensitive profiling of all relevant species and monitoring of their temporal changes under various conditions.
Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.
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