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Normal human dermal fibroblasts: Proteomic analysis of cell layer and culture medium
Author(s) -
Boraldi Federica,
Bini Luca,
Liberatori Sabrina,
Armini Alessandro,
Pallini Vitaliano,
Tiozzo Roberta,
Ronchetti Ivonne Pasquali,
Quaglino Daniela
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200390166
Subject(s) - cell culture , proteome , isoelectric point , gel electrophoresis , two dimensional gel electrophoresis , isoelectric focusing , microbiology and biotechnology , proteomics , chemistry , cell type , cell , biology , biochemistry , genetics , gene , enzyme
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.