title: Fatty-acyl chain profiles of cellular phosphoinositides.
abstract: Phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. Through technical refinements we have improved sensitivity in the analysis of the phosphoinositide PI, PIP, and PIP2pools from living cells using mass spectrometry. This has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. The technique includes butanol extraction, derivatization of the lipids, post-column infusion of sodium to stabilize formation of sodiated adducts, and electrospray ionization mass spectrometry in multiple reaction monitoring mode, achieving a detection limit of 20pg. We describe the spectrum of fatty-acyl chains in the cellular phosphoinositides. Consistent with previous work in other mammalian primary cells, the 38:4 fatty-acyl chains dominate in the phosphoinositides of the pineal gland and of superior cervical ganglia, and many additional fatty acid combinations are found at low abundance. However, Chinese hamster ovary cells and human embryonic kidney cells (tsA201) in culture have different fatty-acyl chain profiles that change with growth state. Their 38:4 lipids lose their dominance as cultures approach confluence. The method has good time resolution and follows well the depletion in <20s of both PIP2and PIP that results from strong activation of Gq-coupled receptors. The receptor-activated phospholipase C exhibits no substrate selectivity among the various fatty-acyl chain combinations.
authors.affiliation: ATK Innovation, Analytics and Discovery, North Bend, WA 98045, USA; Department of Medicine/Gastroenterology, University of Washington School of Medicine, Seattle, WA, USA. Electronic address: atkaplan@uw.edu.
authors.forename: Alexis
authors.initials: A
authors.lastname: Traynor-Kaplan
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Kruse
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Eamonn J
authors.initials: EJ
authors.lastname: Dickson
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Gucan
authors.initials: G
authors.lastname: Dai
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Oscar
authors.initials: O
authors.lastname: Vivas
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Haijie
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Medicinal Chemistry, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Dale
authors.initials: D
authors.lastname: Whittington
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA, USA.
authors.forename: Bertil
authors.initials: B
authors.lastname: Hille
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005227:Fatty Acids / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D010716:Phosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification*; D010743:Phospholipids / Q000737:chemistry / Q000302:isolation & purification*; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D005227:Fatty Acids; D010716:Phosphatidylinositols; D010743:Phospholipids
keywords: Arachidonic acid; Lipidomics; Mass spectrometry; Phospholipids