Freiburg Institute for Advanced Studies, School of Life Science-LifeNet, University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany.
Mol Cell Proteomics. 2013 Sep;12(9):2509-21. doi: 10.1074/mcp.M112.025478. Epub 2013 May 30.
Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes.
角朊细胞占表皮所有细胞的 95%，表皮是形成皮肤外层的复层鳞状上皮，许多皮肤疾病都扎根于此。永生化角朊细胞系常被用作研究模型系统，提供标准化、可重复和同质的生物材料。此外，原代人角质形成细胞常用于医学研究，因为皮肤是药物给药的重要途径，并且很容易进行活检。然而，这些细胞系统的可比性尚不清楚。细胞系可能会经历表型转变，并且在重要方面可能与体内情况不同。另一方面，原代细胞的生物学功能可能因供体的性别和年龄以及活检标本的定位而有所不同。在这里，我们采用代谢标记与基于定量质谱的蛋白质组学相结合的方法，评估 A431 和 HaCaT 细胞系作为模型系统的适用性。与细胞系相比，对原代人角质形成细胞蛋白质组进行全面的性别、年龄和皮肤定位分析，确定了出人意料的高蛋白质组一致性。通过设计用于研究全局蛋白质组学实验的改进本体论富集分析工作流程对数据进行分析。它可以快速、全面和无偏地概述改变的生物学现象，并将实验数据与文献联系起来。我们将指导您完成工作流程，指出与其他方法相比的优势，并应用它来可视化细胞系与原代人角质形成细胞的差异。
