Primary skin fibroblasts as human model system for proteome analysis

Abstract

Elucidation of cellular processes and their changes at the level of protein expression and post-translational modification patterns may allow identification of novel proteins and thereby mechanisms involved in the pathogenesis of multigenic diseases. The aim of this study was to test cultured, nontransformed primary fibroblasts derived from human skin biopsies as a suitable model system for proteome analysis. Therefore soluble protein fractions were separated on several overlapping ultrazoom gels covering the pH range from 3.5-9. Correlation analysis of gel-pairs revealed a highly reproducible protein expression pattern within (intra-assay) and between (inter-assay) independent experiments of a single fibroblast cell line (intra-cell line comparison). Spot intensity variations were less than a factor of two for more than 80% of identical spots. In addition, inter-cell line comparison exhibits no significant variations in spot intensities. To achieve further improvements in reproducibility we generated master gels for each pH range by combining averaged spot information derived from two different cell lines each analysed by two independent experiments using the raw master gel algorithm of the Z3 image analysis software. The resulting reference images of primary human fibroblasts provided a basis for investigating regulation by extracellular stimuli and drugs as well as their alterations in patients with different diseases.

Abnormal cytoskeletal protein expression in cultured skin fibroblasts from type 1 diabetes mellitus patients with nephropathy: A proteomic approach

“…Using 2-DE coupled to MS, differentially expressed proteins among the groups were identified. Correlation analysis of gel-pairs revealed a highly reproducible protein expression pattern both within (intra-assay) and between (interassay) independent experiments, with an average matching efficiency of <75%, a good figure according to a published report [28]. All the analyzed protein spots were found in each subjects' group.…”

The Effects of Rosiglitazone and High Glucose on Protein Expression in Endothelial Cells

“…After spot matching, the following parameters were taken into account to estimate gel-to-gel reproducibility: the efficiency of matching , defined as the percentage of matched spot on the total of detected spots between two gels, a figure that indicates qualitative differences among gels; and the coefficient of variation of matched spot intensities, calculated on a total of 9 samples for each experimental group (see above), that reflects the quantitative differences. These correlation analyses revealed reproducible protein patterns with a an average matching efficiency of 75%, and coefficient of variation of spot intensity of 28%, good figures also in agreement with a published report (see Supplementary Data: Tables 1 and 2 and Figures 1 and 2).…”

Plasticity of protein expression during culture of fetal skin cells

“…2-D gel electrophoresis and proteomics studies of keratinocytes [24][25][26][27][28][29][30] as well as of skin fibroblats [31] has been an area of research that rapidly expanded over the last Ubiquitin-protein hydrolase is involved both in the processing of ubiquitin precursors and of ubiquinated proteins. This enzyme is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin.…”