Bausch-Fluck Damaris, Hofmann Andreas, Wollscheid Bernd
Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
Methods Mol Biol. 2012;909:1-16. doi: 10.1007/978-1-61779-959-4_1.
Proteins expressed at the cell surface define how cells can functionally interact with their microenvironment in time and space. The cell surface subproteome, or surfaceome, represents a cellular information gateway not only enabling the processing of environmental molecular cues but also limiting cellular interaction capacities. Therefore, the array of antibody-detectable cell surface proteins is widely used to phenotype and categorize cells. Quantitative differences in surfaceome markers can not only indicate different developmental cellular stages but also serve as markers of disease. In fact, cell surface proteins are promising biomarker candidates, since they are often, apart from their plasma membrane expression, secreted, shed, or released otherwise from the tissue into the bloodstream. From minute amounts of blood these informative proteins can be detected and quantified by ELISA or highly sensitive state-of-the art targeted mass spectrometric techniques. However, the identification of the complete surfaceome and its constituents is hampered by a lack of suitable technologies to detect these proteins at the cell surface location. Antibodies for the detection of cell surface proteins are only available for a subset of the potentially expressed surfaceome members. The mass spectrometry-based cell surface capturing (CSC) technology and recently developed variants overcome these limitations by selectively enriching and identifying cell surface proteins that are either N-glycosylated (Glyco-CSC, Cys-Glyco-CSC), or have an extracellularly exposed and conformationally available lysine (Lys-CSC). Here, we outline the CSC technology and its variants in a detailed step-by-step protocol for soluble and adherent cells. Representative results from the application of the CSC technologies to the hepatocyte cell line Hepa1-6 illustrate the complementary nature of the CSC technologies, which enables a systems biology view of the surfaceome.
细胞表面表达的蛋白质决定了细胞如何在时间和空间上与微环境进行功能相互作用。细胞表面亚蛋白质组，即表面蛋白质组，代表了一个细胞信息门户，不仅能够处理环境分子信号，还能限制细胞的相互作用能力。因此，一系列可通过抗体检测的细胞表面蛋白质被广泛用于细胞表型分析和分类。表面蛋白质组标记物的定量差异不仅可以指示细胞不同的发育阶段，还可作为疾病的标记物。事实上，细胞表面蛋白质是很有前景的生物标志物候选物，因为除了在质膜上表达外，它们还经常被分泌、脱落或以其他方式从组织释放到血液中。通过酶联免疫吸附测定（ELISA）或高灵敏度的先进靶向质谱技术，可以从微量血液中检测和定量这些信息丰富的蛋白质。然而，由于缺乏在细胞表面位置检测这些蛋白质的合适技术，完整表面蛋白质组及其成分的鉴定受到了阻碍。用于检测细胞表面蛋白质的抗体仅针对潜在表达的表面蛋白质组成员中的一部分。基于质谱的细胞表面捕获（CSC）技术及其最近开发的变体通过选择性富集和鉴定N-糖基化（糖基化CSC、半胱氨酸-糖基化CSC）或具有细胞外暴露且构象可用赖氨酸（赖氨酸CSC）的细胞表面蛋白质，克服了这些限制。在这里，我们以详细的分步方案概述了针对可溶性细胞和贴壁细胞的CSC技术及其变体。将CSC技术应用于肝细胞系Hepa1-6的代表性结果说明了CSC技术的互补性，这使得能够从系统生物学角度看待表面蛋白质组。
