Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry.
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.
Wenk
Markus R
MR
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
Lucast
Louise
L
Di Paolo
Gilbert
G
Romanelli
Anthony J
AJ
Suchy
Sharon F
SF
Nussbaum
Robert L
RL
Cline
Gary W
GW
Shulman
Gerald I
GI
McMurray
Walter
W
De Camilli
Pietro
P
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*
D002457:Cell Extracts; D008055:Lipids; D010716:Phosphatidylinositols