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Multiplexed Lipid Arrays of Anti-Immunoglobulin M-Induced Changes in the Glycerophospholipid Composition of WEHI-231 Cells
Department of Pharmacology and the Institute for Chemical Biology, Vanderbilt University Medical Center, Nashville, TN
Abstract
A goal of the Alliance for Cellular Signaling (AfCS) is to identify the diverse participants that compose an intracellular signaling network. Phospholipids are important participants in transmembrane signaling processes as well as direct mediators of the dynamic aspects of cell membrane structure. Therefore, identifying the contextual changes in membrane lipid composition (in addition to that of genes and proteins) is essential in achieving a comprehensive understanding of signaling networks in cells. Recent advances in high-throughput electrospray ionization mass spectroscopy (ESI-MS) coupled with new computational approaches have greatly facilitated this goal. One cell type of interest to the AfCS is the splenic B lymphocyte and its experimental surrogate, the WEHI-231 cell (1, 2). Here we identify more than 200 species of glycerophospholipids from total membrane extracts of WEHI-231 cells and qualitatively measure pattern response changes initiated by stimulation of cell surface receptors.
In these studies, WEHI cells were treated with anti-immunoglobulin M antibody (AIG) to stimulate the B-cell receptor. The response to AIG stimulation was a conspicuous change in a broad range of phospholipids. An overall temporal trend was observed in which lipid concentration changes were detected by 6 minutes, pattern changes peaked by 15 minutes, and by 4 hours of stimulation, the cells had largely returned to their prestimulated composition. Statistically significant decreases were observed in many species of phospholipids along with concomitant increases in lysophospholipid concentrations. This study represents the most comprehensive analysis of membrane phospholipid changes in any cell type to date. The procedure described can be applied to any mammalian cell type and provides a basis for the comprehensive study of lipid signal transduction. Taken together, these changes form unique patterns that will be used to discriminate ligand-stimulated events and to model signaling pathways that lead to developmental and phenotypic changes in cells.
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