Journal
NATURE METHODS
Volume 8, Issue 3, Pages 267-U120Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1564
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Funding
- Biotechnology and Biological Sciences Research Council
- European Union [202272]
- BBSRC [BBS/E/B/0000C218, BBS/E/B/0000H213, BB/I003916/1, BB/H024824/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/B/0000C218, BB/H024824/1, BB/I003916/1, BBS/E/B/0000H213] Funding Source: researchfish
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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.
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