Abstract
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.
Highlights
The epidermis is the outermost layer of the skin protecting the entire organism against insults from the external environment and providing an impenetrable barrier against fluid and electrolyte loss [1]
A Bioinformatics Workflow for Hypothesis Free, Global Proteomics Studies—We designed a five-step workflow for the unbiased, comprehensive analysis of “omics” data, in which we implemented four improvements compared with classical ontology enrichment analyses
We provide a comprehensive overview of the keratinocyte proteome and explore proteomic alterations in keratinocytes of different origin that are commonly used as model systems
Summary
The epidermis is the outermost layer of the skin protecting the entire organism against insults from the external environment and providing an impenetrable barrier against fluid and electrolyte loss [1]. To study proteome alterations of primary human keratinocytes and to evaluate the suitability of keratinocyte-like cell line models we conducted an unbiased global mass spectrometry (MS)-based proteome analysis Such protein abundance analyses are usually combined with ontology enrichment tests to gain an unbiased and comprehensive overview of altered biological functions. We introduce a sliding window ontology enrichment analysis that combines the advantages of the over-representation approach and the aggregate score approach and visualizes significance of over-representation for different extents of regulation. It is the core of a five-step bioinformatics data processing workflow designed to provide a quick and comprehensive overview of altered biological phenomena in unbiased, global proteomics experiments. To the comparison to HaCaT cells we investigated the influence of age, gender, and skin localization on the proteome of primary human keratinocytes
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