BET CELLS & MASS SPECTROMETERS

I型糖尿病和II型糖尿病都是由于 胰岛中分泌胰岛素的β细胞的功能或丧失。 β细胞膜控制离子电导、胞吐作用， 抗原特性和复杂的脂质成分影响膜 功能协调发展的 胰岛脂质的表征是困难的， 胰岛只能少量获得。 常规方法 复杂的脂质分析涉及多个色谱，消化， 和衍生化步骤，每个步骤都涉及分析物损失，以及FAB/MS 灵敏度不足以允许分析 胰岛亚细胞膜。 ESI/MS联用灵敏度高， 由碰撞激活的 解离和串联质谱法避免了这些 困难 我们开发了ESI/MS/MS方法， 少量甘油磷酸胆碱脂质的鉴定， 硫苷脂种类。 ESI/MS已用于鉴定所有主要的 胰岛分泌颗粒和质膜中的磷脂种类， 以鉴定胰岛硫脂种类，并检查刺激诱导的 胰岛磷脂种类数量的变化。 表征 胰岛脂质导致发现了一种新的脂质代谢 这种酶现在被命名为iPLA2。 我们过度表达了 Sf9细胞中的酶，并表征其翻译后 胰蛋白酶消化物的HPLC/ESI/MS/MS分析的修改 重组蛋白

Both types I and II diabetes mellitus result from abnormal function or loss of insulin-secreting beta cells in pancreatic islets. Beta cell membranes govern ionic conductances, exocytosis, and antigenic properties, and complex lipid components affect membrane functions. Characterization of islet lipids is difficult because islets are obtainable in only small quantities. Conventional methods of complex lipid analysis involve multiple chromatographic, digestion, and derivatization steps that each involve analyte losses, and FAB/MS is insufficiently sensitive to permit analysis of complex lipids in islet subcellular membranes. The great sensitivity of ESI/MS combined with structural information provided by collisionally activated dissociation and tandem mass spectrometry circumvents these difficulties. We have developed ESI/MS/MS methods that facilitate identification of small quantities of glycerophosphocholine lipids and of sulfatide species. ESI/MS has been used to identify a ll major phospholipid species in islet secretory granules and plasma membranes, to identify islet sulfatide species, and to examine stimulus-induced changes in amounts of islet phospholipid species. Characterization of islet lipids has led to the discovery of a new lipid metabolizing enzyme, which is now designated iPLA2. We have overexpressed this enzyme in Sf9 cells and are characterizing its post-translational modifications by HPLC/ESI/MS/MS analyses of tryptic digetsts of the recombinant protein.