title: Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.
abstract: Phosphoinositides (phosphorylated derivatives of phosphatidylinositol, PI) are versatile intracellular signaling lipids whose occurrence in low concentrations complicates direct mass measurements. Here we present a sensitive method to detect, identify and quantify phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)) with different fatty acid compositions (phosphoinositide profiles) in total lipid extracts by electrospray ionization mass spectrometry (ESI-MS). Using this method, we detected elevated concentrations of PIP2 in human fibroblasts from patients with Lowe syndrome, a genetic disorder that affects phosphoinositide metabolism. Saccharomyces cerevisiae cells deficient in enzymes involved in PIP metabolism--Sac1p, a phosphoinositide phosphatase, and Vps34p and Pik1p, a PI 3-kinase and PI 4-kinase, respectively--showed not only different PIP concentrations but also differential changes in PIP profiles indicating metabolic and/or subcellular pooling. Mass spectrometric analysis of phosphoinositides offers unique advantages over existing approaches and may represent a powerful diagnostic tool for human diseases that involve defective phosphoinositide metabolism.
authors.affiliation: Howard Hughes Medical Institute, Department of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, Connecticut 06511, USA. markus.wenk@yale.edu
authors.forename: Markus R
authors.initials: MR
authors.lastname: Wenk
authors.forename: Louise
authors.initials: L
authors.lastname: Lucast
authors.forename: Gilbert
authors.initials: G
authors.lastname: Di Paolo
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Romanelli
authors.forename: Sharon F
authors.initials: SF
authors.lastname: Suchy
authors.forename: Robert L
authors.initials: RL
authors.lastname: Nussbaum
authors.forename: Gary W
authors.initials: GW
authors.lastname: Cline
authors.forename: Gerald I
authors.initials: GI
authors.lastname: Shulman
authors.forename: Walter
authors.initials: W
authors.lastname: McMurray
authors.forename: Pietro
authors.initials: P
authors.lastname: De Camilli
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002457:Cell Extracts / Q000737:chemistry*; D002478:Cells, Cultured; D005240:Feasibility Studies; D008055:Lipids / Q000032:analysis* / Q000737:chemistry*; D051379:Mice; D009474:Neurons / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000032:analysis* / Q000737:chemistry*; D051381:Rats; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D002457:Cell Extracts; D008055:Lipids; D010716:Phosphatidylinositols