Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation

Abstract

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A 2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono-and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis. mast cells ͉ phospholipases ͉ exocytosis P hospholipids play important roles in transmembrane signaling processes as well as in dynamic aspects of cell membrane structure. The major phospholipids found in the membranes of mammalian cells include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG). Some exist as both sphingolipids and glycerophospholipids, as plasmalogens and glycerol diesters, as polyphosphorylated species (e.g., PIs), and with a diverse and specialized array of alkyl chains. Phospholipid metabolism is regulated by distinct types of extracellular receptor-regulated pathways in a variety of ways, including generation of first and second messengers [inositol trisphosphate, phosphatidylinositol 4,5-bisphosphate, platelet-activating factor, diacylglycerol (DAG), sphingosine, etc.]; modifications associated with membrane fusion, secretion, trafficking, and plasma membrane shape change; and to date vaguely described changes in bilayer structure needed to regulate the activities of enzymes, channels, and transport proteins. The chemical and physical properties of membranes are largely dependent on the phospholipid composition (1-4). Although the biological significance of lipid heteroge...

FcϵRI and Thy-1 domains have unique protein and lipid compositions

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Lipid Profiling Reveals Arachidonate Deficiency in RAW264.7 Cells: Structural and Functional Implications

“…The recent development of mass spectrometric (MS) techniques for the detection and analysis of total cellular lipids allows the detailed examination of the glycerophospholipid structure and composition of cells (19,20), providing an unprecedented opportunity to evaluate the comprehensive effects of cellular signaling events on the entire metabolome. Here we apply these techniques to a primary macrophage population, RPM, which has been extensively studied as a source of oxygenated 20:4 metabolites (10)(11)(12)(13)(14)(15)(16), and the RAW264.7 murine macrophage cell line (21), which is frequently used as a model for primary macrophages.…”

Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcϵRI

“…These lipases generate important lipid second messengers, as well as amphiphilic products that can modulate membrane structure and influence processes such as vesicle budding and exocytosis (Liscovitch et al, 2000;Cohen and Brown, 2001;Ivanova et al, 2001). Ceramides are the products of sphingomyelin hydrolysis by sphingomyelinases, and these endogenous, long-chain neutral lipids have been implicated as signal transduction mediators in certain cell types (Kolesnick et al, 2000).…”