Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)

• Published in: The Protein Journal Vol. 37; no. 6; pp. 589 - 607
• Main Authors: Maarof, Manira, Lokanathan, Yogeswaran, Ruszymah, Hj Idrus, Saim, Aminuddin, Chowdhury, Shiplu Roy
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-12-2018; Springer; Springer Nature B.V
• Subjects: Animal Anatomy; Biochemistry; Bioinformatics; Biological activity; Bioorganic Chemistry; Care and treatment; Cell adhesion; Cell adhesion & migration; Cell culture; Chemistry; Chemistry and Materials Science; Collagen; Comparative analysis; Conditioning; Culture media; Electrophoresis; Environmental factors; Enzyme-linked immunosorbent assay; Ethylenediaminetetraacetic acid; Extracellular matrix; Fibroblasts; Fibronectin; Gel electrophoresis; Gene expression; Growth factors; Histology; In vivo methods and tests; Ionization; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Morphology; Organic Chemistry; Proteins; Serum albumin; Serum-free medium; Skin; Sodium dodecyl sulfate; Sodium lauryl sulfate; Statistical analysis; Wound healing; Wounds and injuries; Dermal fibroblast; Wound healing; Conditioned medium; Secretory protein
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-018-9800-z

Growth factors and extracellular matrix (ECM) proteins are involved in wound healing. Human dermal fibroblasts secrete wound-healing mediators in culture medium known as dermal fibroblast conditioned medium (DFCM). However, the composition and concentration of the secreted proteins differ with culture conditions and environmental factors. We cultured human skin fibroblasts in vitro using serum-free keratinocyte-specific media (EpiLife™ Medium [KM1] and defined keratinocyte serum-free medium [KM2]) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. We identified and compared their proteomic profiles using bicinchoninic acid assay (BCA), 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography MS (LC-MS/MS). DFCM-KM1 and DFCM-KM2 had higher protein concentrations than DFCM-FM but not statistically significant. MALDI-TOF/TOF MS identified the presence of fibronectin, serotransferrin, serpin and serum albumin. LC-MS/MS and bioinformatics analysis identified 59, 46 and 58 secreted proteins in DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. The most significant biological processes identified in gene ontology were cellular process, metabolic process, growth and biological regulation. STRING® analysis showed that most secretory proteins in the DFCMs were associated with biological processes (e.g. wound healing and ECM organisation), molecular function (e.g. ECM binding) and cellular component (e.g. extracellular space). ELISA confirmed the presence of fibronectin and collagen in the DFCMs. In conclusion, DFCM secretory proteins are involved in cell adhesion, attachment, proliferation and migration, which were demonstrated to have potential wound-healing effects by in vitro and in vivo studies.