Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)

Manira Maarof, Lokanathan Yogeswaran, Hj Idrus Ruszymah, Aminuddin Saim, Shiplu Roy Chowdhury

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)589-607
Number of pages19
JournalProtein Journal
Volume37
Issue number6
DOIs
Publication statusPublished - 1 Dec 2018

Fingerprint

Fibroblasts
Conditioned Culture Medium
Proteomics
Skin
Proteins
Biological Phenomena
Wound Healing
Assays
Immunosorbents
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Keratinocytes
Fibronectins
Extracellular Matrix
Enzymes
Enzyme-Linked Immunosorbent Assay
Serpins
Gene Ontology
Extracellular Matrix Proteins
Cell adhesion
Serum-Free Culture Media

Keywords

  • Conditioned medium
  • Dermal fibroblast
  • Secretory protein
  • Wound healing

ASJC Scopus subject areas

  • Analytical Chemistry
  • Bioengineering
  • Biochemistry
  • Organic Chemistry

Cite this

Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM). / Maarof, Manira; Yogeswaran, Lokanathan; Ruszymah, Hj Idrus; Saim, Aminuddin; Chowdhury, Shiplu Roy.

In: Protein Journal, Vol. 37, No. 6, 01.12.2018, p. 589-607.

Research output: Contribution to journalArticle

Maarof, Manira ; Yogeswaran, Lokanathan ; Ruszymah, Hj Idrus ; Saim, Aminuddin ; Chowdhury, Shiplu Roy. / Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM). In: Protein Journal. 2018 ; Vol. 37, No. 6. pp. 589-607.
@article{c02910675f0243a18191819ed9ccdf6e,
title = "Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)",
abstract = "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{\circledR} 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.",
keywords = "Conditioned medium, Dermal fibroblast, Secretory protein, Wound healing",
author = "Manira Maarof and Lokanathan Yogeswaran and Ruszymah, {Hj Idrus} and Aminuddin Saim and Chowdhury, {Shiplu Roy}",
year = "2018",
month = "12",
day = "1",
doi = "10.1007/s10930-018-9800-z",
language = "English",
volume = "37",
pages = "589--607",
journal = "Protein Journal",
issn = "1572-3887",
publisher = "Springer New York",
number = "6",

}

TY - JOUR

T1 - Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)

AU - Maarof, Manira

AU - Yogeswaran, Lokanathan

AU - Ruszymah, Hj Idrus

AU - Saim, Aminuddin

AU - Chowdhury, Shiplu Roy

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

AB - 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.

KW - Conditioned medium

KW - Dermal fibroblast

KW - Secretory protein

KW - Wound healing

UR - http://www.scopus.com/inward/record.url?scp=85055690308&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85055690308&partnerID=8YFLogxK

U2 - 10.1007/s10930-018-9800-z

DO - 10.1007/s10930-018-9800-z

M3 - Article

VL - 37

SP - 589

EP - 607

JO - Protein Journal

JF - Protein Journal

SN - 1572-3887

IS - 6

ER -