Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization

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. Molecular &

Determination of cell type‐specific proteome signatures of primary human leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts and melanocytes by comparative proteome profiling

“…In order to test also the keratinocyte signature, we searched for literature describing skin proteome profiles, which is rather scarce. However, Sprenger et al have presented an analysis of primary human keratinocytes listing more than 4000 proteins , which is also compatible with our signatures as it contains three members of our keratinocyte signature. Unexpectedly, the other three keratinocyte signature members, which are related to typical keratinocyte functions, are missing in this study.…”

Deep Sequencing MicroRNAs from Extracellular Membrane Vesicles Revealed the Association of the Vesicle Cargo with Cellular Origin

“…However, there are also distinct BP GO terms associated with EV parental cell origin. These disparities may arise from the specific differences in the physiological conditions between the HaCaT cell line, which resemble transformed keratinocytes, and primary keratinocyte cells, that while more biologically relevant exhibit heterogeneity between donors [30,31]. Both the experimental and bioinformatic data indicated that fibroblast migration may be regulated by PKC-derived EVs but not by HaCaT-derived EVs, which demonstrates the association of biological processes with EV cellular origin.…”

Quantitative proteogenomic profiling of epidermal barrier formation in vitro

“…Across all samples in this pilot experiment, 2412 proteins were identified at a 1% FDR, with nearly complete coverage of unfractionated results in the fractionated samples (Figure 1C). Despite analyzing only six samples in this test set, this number of protein identifications in a skin-related system is rivaled only by studies on undifferentiated primary keratinocytes or cell lines [12]. …”

“…To overcome this issue, past efforts have frequently employed separation of proteins via gel electrophoresis [8–10], a procedure with low throughput as well as poor sensitivity for lower abundance proteins [11]. Several published studies have been carried out using relatively undifferentiated cultured cells which do not suffer from such extreme dynamic range [12–15]. Such studies can produce a large number of protein identifications but do not accurately represent the stratified structure and resulting protein profile of natural epidermis.…”