Direct analysis of PI(3,4,5)P3 using liquid chromatography electrospray ionization tandem mass spectrometry

Analytical Biochemistry
Hai H BuiAnthony S Fischl

Abstract

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a biologically active membrane phospholipid that is essential for the growth and survival of all eukaryotic cells. We describe a new method that directly measures PIP3 and describe the HPLC separation and measurement of the positional isomers of phosphatidylinositol bisphosphate, PI(3,5)P2, PI(3,4)P2 and PI(4,5)P2. Mass spectrometric analyses were performed online using ultra-high performance liquid chromatography (UHPLC)-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the negative multiple-reaction monitoring (MRM) modes. Rapid separation of PIP3 from PI, phosphatidylinositol phosphate (PIP) and PIP2 was accomplished by C18 reverse phase chromatography with the addition of the ion pairing reagents diisopropylethanolamine (DiiPEA) and ethylenediamine tetraacetic acid tetrasodium salt dihydrate (EDTA) to the samples and mobile phase with a total run time, including equilibration, of 12 min. Importantly, these chromatography conditions result in no carryover of PIP, PIP2, and PIP3 between samples. To validate the new method, U87MG cancer cells were serum starved and treated with PDGF to stimulate PIP3 biosynthesis in the presence or absence of the phosphatidy...Continue Reading

Citations

Nov 6, 2018·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Yoshibumi UedaMoritoshi Sato
Oct 28, 2019·Biochemical Society Transactions·David BarnedaPhillip Hawkins
Dec 29, 2018·The Journal of Cell Biology·Brady D GouldenGerald R V Hammond
May 7, 2020·Journal of Lipid Research·Koralege C PathmasiriStephanie M Cologna
Apr 8, 2020·Cells·Theodore G Wensel
Nov 20, 2019·Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids·Tom N DurrantIngeborg Hers
Feb 23, 2021·Frontiers in Cell and Developmental Biology·Fernando Montaño-RendónGlenn F W Walpole
May 10, 2021·Bioorganic & Medicinal Chemistry·Alex G Batrouni, Jeremy M Baskin

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