Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry
Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry
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DOI：
10.1021/bi9828324
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发表时间：
1999-06-22
期刊：
影响因子：
2.9
通讯作者：
McLafferty, FW
中科院分区：
文献类型：
--
作者：
Fridriksson, EK;Shipkova, PA;McLafferty, FW
我们最近表明，在RBL-2 H3肥大细胞上，免疫球蛋白E的高亲和性受体Fc ε RI的配体介导的交联导致其与去污剂抗性膜（DRM）的共分离以及其随后通过共定位的酪氨酸激酶林恩的酪氨酸磷酸化，这是通过该受体进行信号传导的关键早期事件[Field等人（1997）J.Biol.Chem.272，4276-4280]。作为确定这些相互作用的结构基础的努力的一部分，我们检查了在保留Fc γ RT结合的条件下从RBL-2 H3细胞分离的DRM囊泡的磷脂组成。我们使用正和负模式电喷雾傅立叶变换离子回旋共振质谱定量比较分离DRM的磷脂组合物的总细胞脂质和质膜制备。从这些分析中，超过90种不同的磷脂物质进行了光谱解析和明确确定，其中超过三分之二的测定精度为+/-0.5%（绝对值）或更低。脂质谱的定量表征表明，分离的DRM基本上富含鞘磷脂和甘油磷脂，与总细胞相比：脂质具有更高的饱和度。通过化学诱导起泡从RBL-2 H3细胞分离的质膜囊泡表现出一定程度的磷脂饱和度，其介于DRM和总细胞脂质之间，并且观察到DRM和质膜囊泡之间的头基分布的显著差异。来自具有交联Fc β RI的细胞的DRM比来自未刺激的细胞的DRM表现出更大的多不饱和磷脂与饱和磷脂和单不饱和磷脂的比率。我们的研究结果支持并加强了以前的研究结果，表明DRM具有促进液体有序结构的脂质成分。此外，它们还证明了质谱法用于检查膜结构在受体信号传导和其他细胞过程中的作用的潜力。
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 consequent tyrosine phosphorylation by the co-localized tyrosine kinase Lyn that is a critical early event in signaling by this receptor [Field et al. (1997) J. Biol. Chem. 272, 4276-4280]. As part of efforts to determine the structural bases for these interactions, we examined the phospholipid composition of DRM vesicles isolated from RBL-2H3 cells under conditions that preserve Fc epsilon RT. association. We used positive and negative mode electrospray Fourier transform ion cyclotron resonance mass spectrometry to compare quantitatively the phospholipid composition of isolated DRM to that of total cell lipids and to a plasma membrane preparation. From these analyses, over 90 different phospholipid species were spectrally resolved and unambiguously identified; more than two-thirds of these were determined with a precision of +/-0.5% (absolute) or less. Quantitative characterization of lipid profiles shows that isolated DRM are substantially enriched in sphingomyelin and in glycerophospholipids with a higher degree of saturation as compared to total cellular: lipids. Plasma membrane vesicles isolated from RBL-2H3 cells by chemically induced blebbing exhibit a degree of phospholipid saturation that is intermediate between DRM and total cellular lipids, and significant differences in the headgroup distribution between DRM and plasma membranes vesicles are observed. DRM from cells with cross-linked Fc epsilon RI exhibit a larger ratio of polyunsaturated to saturated and monounsaturated phospholipids than those from unstimulated cells. Our results support and strengthen results from previous studies suggesting that DRM have a lipid composition that promotes liquid-ordered structure. Furthermore, they demonstrate the potential of mass spectrometry for examining the role of membrane structure in receptor signaling and other cellular processes.