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首页> 外文期刊>The Journal of investigative dermatology. >A Proteomic Characterization of the Plasma Membrane of Human Epidermis by High-Throughput Mass Spectrometry.
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A Proteomic Characterization of the Plasma Membrane of Human Epidermis by High-Throughput Mass Spectrometry.

机译：高通量质谱法对人表皮质膜的蛋白质组学表征

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摘要

Membrane proteins are responsible for many critical cellular functions and identifying cell surface proteins on different keratinocyte populations by proteomic approaches would improve our understanding of their biological function. The ability to characterize membrane proteins, however, has lagged behind that of soluble proteins both in terms of throughput and protein coverage. In this study, a membrane proteomic investigation of keratinocytes using a two-dimensional liquid chromatography (LC) tandem-mass spectrometry (MS/MS) approach that relies on a buffered methanol-based solubilization, and tryptic digestion of purified plasma membrane is described. A highly enriched plasma membrane fraction was prepared from newborn foreskins using sucrose gradient centrifugation, followed by a single-tube solubilization and tryptic digestion of membrane proteins. This digestate was fractionated by strong cation-exchange chromatography and analyzed using microcapillary reversed-phase LC-MS/MS. In a set of 1306 identified proteins, 866 had a gene ontology (GO) annotation for cellular component, and 496 of these annotated proteins (57.3%) were assigned as known integral membrane proteins or membrane-associated proteins. Included in the identification of a large number of aqueous insoluble integral membrane proteins were many known intercellular adhesion proteins and gap junction proteins. Furthermore, 121 proteins from cholesterol-rich plasma membrane domains (caveolar and lipid rafts) were identified.
机译：膜蛋白负责许多关键的细胞功能，通过蛋白质组学方法鉴定不同角质形成细胞群上的细胞表面蛋白将增进我们对其生物学功能的理解。然而，就通量和蛋白质覆盖率而言，表征膜蛋白的能力均落后于可溶性蛋白。在这项研究中，描述了使用二维液相色谱（LC）串联质谱（MS / MS）方法对角质形成细胞进行膜蛋白质组学研究，该方法依赖于基于甲醇的缓冲增溶作用，并对纯化的质膜进行胰蛋白酶消化。使用蔗糖梯度离心法，从新生的包皮中制备高度浓缩的质膜级分，然后单管溶解并通过胰蛋白酶消化膜蛋白。通过强阳离子交换色谱分离该消化物，并使用微毛细管反相LC-MS / MS进行分析。在一组1306个已鉴定的蛋白质中，有866个具有细胞本体的基因本体（GO）注释，其中496个（57.3％）的注释蛋白质被指定为已知的整合膜蛋白质或膜相关蛋白质。在鉴定大量水不溶性整体膜蛋白时，包括许多已知的细胞间粘附蛋白和间隙连接蛋白。此外，鉴定了来自富含胆固醇的质膜结构域（肺泡和脂质筏）的121种蛋白质。

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