Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence at least one energetic pyrophosphate moiety on myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, cellular energy charge. Owing to absence a chromophore metabolites, very high charge density, low abundance, their analysis requires radioactive tracer, thus it is convoluted expensive. Here, study presents detailed protocol perform absolute throughput quantitation mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables sensitive profiling all biologically relevant PP-InsPs species cells, enabling baseline separation regioisomers. Absolute concentrations PP-InsPs, including minor isomers, monitoring temporal changes HCT116 under several experimental conditions presented.