Background: The primary cell of the gingiva and periodontal ligament, the fibroblast, mainly functions to provide tissue homeostasis, integrity, communication, and immunity. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) exhibit several similarities, such as proliferation, migration, collagen synthesis, phagocytosis, expression of non-collagenous proteins, and outward appearance. However, many differences do exist between GF and PDLF and have been noted in culture, whether based on proliferative capacity and glycosaminoglycan content, chemotactic response, protein and collagen synthesis, or capacity to form mineralized tissue. Explanation for the differences in behavior and content of these cells may be evident in the proteome of the two cell types. The purpose of this study was to describe and compare the insoluble, membrane bound and associated proteins of GF and PDLF.
Methods: GF and PDLF were established from non-inflamed tissue of four systemically healthy subjects grown in cell culture consisting of MEM supplemented with 10% FBS. GF and PDLF were harvested in the 4th passage and membrane bound and associated proteins were isolated from fibroblasts. The protein samples were submitted for enzyme digestion and identification using liquid chromatography/ tandem mass spectrometry. Samples were separated on a capillary column using an UltiMate¿¿¿¿¿¿¿ 3000 HPLC system from LC-Packings A Dionex Co (Sunnyvale, CA). Each sample was injected into the Precolumn Cartridge (Dionex, Sunnyvale, CA) and desalted with 50 mM acetic acid. Mobile phase A was 0.1% formic acid in water and 0.1% formic acid in acetonitrile was used as mobile phase B. Flow rate was set at 2µl/min. MS/MS data was acquired with a spray voltage of 2 KV at a capillary temperature of 175°C. A MS/MS scan generated product ion spectra to determine amino acid sequence. The mass accuracy of the precursor ions were set to 2.0 Da given that the data was acquired on an ion trap mass analyzer and the fragment mass accuracy was set to 0.5 Da. Considered modifications (variable) were methionine oxidation and carbamidomethyl cysteine. Scaffold (Proteome Software, Portland, Oregon, USA) was used to validate protein identifications derived from MS/MS sequencing results. Differences in proteins were considered statistically significant when means were at or greater than a 95% confidence interval.
Results: A total of five hundred nineteen proteins were identified from the eight samples analyzed via LC-MS/MS. Four hundred fifty proteins were common to both GF and PDLF. Forty
proteins were unique to GF and 29 were unique to PDLF. Of the proteins identified two hundred thirteen are known membrane bound or associated proteins. Twenty-eight proteins, identified from the 450 proteins common to both GF and PDLF, were detected in statistically significant greater quantities by either GF or PDLF. Five membrane proteins were detected in greater quantities by GF, while seven membrane proteins were detected in greater quantities by PDLF. Nineteen and ten membrane proteins were identified only in GF and PDLF respectively.
Conclusion: The current study is the first report in the literature to use proteomic analysis to identify and quantify the membrane bound and associated proteins of GF and PDLF. Furthermore it is also the first report to compare and contrast GF and PDLF membrane bound and associated proteins. The use of label free analysis allowed for the most inclusive description of the GF and PDLF proteomes for these 4 matched pairs. By exploring the functional characterization of the differences found between GF and PDLF proteomes we can speculate how the descriptive differences reported in this study relate to fibroblast function.