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

Biological membranes contain an extraordinary diversity of lipids. Phosphol ipids function as major structural elements of cellular membranes, and anal ysis of changes in the highly heterogeneous mixtures of lipids found in euk aryotic cells is central to understanding the complex functions in which li pids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that g ranule fusion is initiated by addition of exogenous, nonmammalian phospholi pases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectromet ry (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 effi cient production of molecular ions of phospholipids uncomplicated by furthe r fragmentation, and changes were observed that eluded conventional detecti on methods. From these analyses we have spectrally resolved more than 130 g lycerophospholipids and determined changes initiated by introduction of exo genous phospholipase C, phospholipase D, or phospholipase A(2). These exoge nous phospholipases have a preference for phosphatidylcholine with long pol yunsaturated alkyl chains as substrates and, when added to permeabilized ma st cells, produce multiple species of mono- and polyunsaturated diacylglyce rols, phosphatidic acids. and lysophosphatidylcholines. respectively. The p atterns of changes of these lipids provide an extraordinarily rich source o f data for evaluating the effects of specific lipid species generated durin g cellular processes, such as exocytosis.