Qiu, D; Eisenbeis, VB; Saiardi, A; Jessen, HJ; (2021) Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry. Journal of Visualized Experiments (174) , Article e62847. 10.3791/62847.

Preview

Text Jove Qui.pdf - Accepted Version Download (856kB) | Preview

Abstract

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.

Download activity - last month

Export as XMLJSONCSV

Download activity - last 12 months

Export as XMLJSONCSV

Downloads by country - last 12 months

Export as CSVJSONXML

Archive Staff Only

View Item