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Nat Methods. 2011 Mar;8(3):267-72. doi: 10.1038/nmeth.1564. Epub 2011 Jan 30.

Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry.

Author information

1
Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, UK.

Abstract

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

PMID:
21278744
PMCID:
PMC3460242
DOI:
10.1038/nmeth.1564
[Indexed for MEDLINE]
Free PMC Article

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