Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts.
The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.
Pflieger
Delphine
D
Neurobiologie et Diversité Cellulaire, ESPCI CNRS UMR 7637, Paris, France. delphine.pflieger@univ-evry.fr
Chabane
Sandrine
S
Gaillard
Olivier
O
Bernard
Bruno Alain
BA
Ducoroy
Patrick
P
Rossier
Jean
J
Vinh
Joëlle
J
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D016326:Extracellular Matrix Proteins / Q000032:analysis*; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D018859:Hair Follicle / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D012867:Skin / Q000166:cytology*
D016326:Extracellular Matrix Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D016650:Fluorescein-5-isothiocyanate