Normal human dermal fibroblasts: Proteomic analysis of cell layer and culture medium

Abstract

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

Differential proteomic analysis of proteins induced by glucocorticoids in cultured murine podocytes

“…It is, however, difficult to draw any conclusions from the relative paucity of proteins common to the podocyte proteome described in this analysis and other currently available kidney proteomes analyses, primarily because the published renal proteomes consist only of proteins differentially expressed in response to some stimulus [36] or common in both cortical and medullary tissues [33,37]. Proteomic analyses of renal cell carcinomas [30,31], cultured fibroblasts [38], umbilical vein endothelial cells [39], and, in particular, cultured, differentiated mammary cells [40] contained a larger and more diverse subset of proteins in common with cultured, differentiated podocytes. The greater overlap between known proteomes of cultured cells than between cultured podocytes and whole kidney is not particularly noteworthy, particularly since the analyses of renal tissues were restricted to a small subset of identified proteins.…”

Age-related decline in expression of calnexin

“…Although blockade of the coronary vasculature is known as a common cause of damage to myocardium, other changes in cellular level that occur during the aging process result in decreasing function of myocardium and rendering it more susceptible to damages (Verbeke et al, 2001;Taylor and Starnes, 2003). Several reports have already described a decreased capacity of the aging myocardium to different types of stresses (Gray et al, 2000;Verbeke et al, 2001;Boraldi et al, 2003;Taylor and Starnes, 2003). Furthermore, age-related alterations in the expression and activation of proteins are known to be important for the cardioprotective process such as heat shock protein (HSP) 70 (Taylor and Starnes, 2003).…”

Proteomic Analysis of the Soluble Fraction from Human Corneal Fibroblasts with Reference to Ocular Transparency

“…Most of the identified proteins are housekeeping proteins common to most other cell types from nontransparent tissues. Thus, a significant number of the identified proteins are also expressed by other fibroblastic cells such as dermal fibroblasts (48,49) and MRC5 fibroblasts (50) (visit proteomics.cancer.dk/jecelis/human_data_select.html). This finding may reflect a common mechanism of light scattering from reflective cells.…”