Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry

We recently showed that ligand-mediated cross-linking of Fc epsilon RI, the high-affinity receptor for immunoglobulin E, on RBL-2H3 mast cells results in its co-isolation with detergent-resistant membranes (DRM) and its conse quent tyrosine phosphorylation by the co-localized tyrosine kinase Lyn that is a critical early event in signaling by this receptor [Field et al. (199 7) J. Biol. Chem. 272, 4276-4280]. As part of efforts to determine the stru ctural bases for these interactions, we examined the phospholipid compositi on of DRM vesicles isolated from RBL-2H3 cells under conditions that preser ve Fc epsilon RT. association. We used positive and negative mode electrosp ray Fourier transform ion cyclotron resonance mass spectrometry to compare quantitatively the phospholipid composition of isolated DRM to that of tota l cell lipids and to a plasma membrane preparation. From these analyses, ov er 90 different phospholipid species were spectrally resolved and unambiguo usly identified; more than two-thirds of these were determined with a preci sion of +/-0.5% (absolute) or less. Quantitative characterization of lipid profiles shows that isolated DRM are substantially enriched in sphingomyeli n and in glycerophospholipids with a higher degree of saturation as compare d to total cellular: lipids. Plasma membrane vesicles isolated from RBL-2H3 cells by chemically induced blebbing exhibit a degree of phospholipid satu ration that is intermediate between DRM and total cellular lipids, and sign ificant differences in the headgroup distribution between DRM and plasma me mbranes vesicles are observed. DRM from cells with cross-linked Fc epsilon RI exhibit a larger ratio of polyunsaturated to saturated and monounsaturat ed phospholipids than those from unstimulated cells. Our results support an d strengthen results from previous studies suggesting that DRM have a lipid composition that promotes liquid-ordered structure. Furthermore, they demo nstrate the potential of mass spectrometry for examining the role of membra ne structure in receptor signaling and other cellular processes.