Background: The role of cells derived from the periodontal ligament has always been considered an important factor in the regeneration of the periodontium. Although the genome for the periodontal ligament fibroblast (PDLF) and gingival fibroblast (GF) is the same in any individual, there have been distinct biochemical differences identified between the two cell types. The differences in behavior of these two cells, despite similar DNA content, may be explained by proteomics, specifically via the proteome of their nuclear membrane, since the nuclear membrane is arguably one of the most essential cellular organelles involved in biogenesis. The principal aim of this study was to determine if there were differences in protein content of enriched nuclear membranes from PDLF and GF.
Methods: PDLF and GF were derived from non-inflamed tissue of 3 systemically healthy subjects, and explants were grown in cell culture consisting of MEM supplemented with 10% FBS. PDLF and GF whole cell suspensions were lysed and nuclear pellets were separated via differential centrifugation using sucrose gradients until an enriched nuclear pellet was present. Samples were cleaned and quantified by DC protein quantitation assay. The same amount of protein (15 micrograms) was taken from each sample evaluated. In addition, 2.5 micrograms of protein was taken from each sample, respectively, and combined as an internal standard. Samples were digested with trypsin, followed by iTRAQ™ mass labeling for peptide qualitative and quantitative identification following capillary LC-MS/MS based proteomic methods for comparative analysis. Missed cleavage number was set at 4. Mass tolerance for peptides was set at 1.2Da and 0.8Da for MS/MS fragments. MASCOT provided qualitative results, and quantitative results were reported as the average ratios between the sample and internal standard. LC-MS/MS was performed multiple times, and the average ratios of protein for matched sets of GF versus PDLF were calculated with standard deviations. Differences in proteins were considered statistically significant when means were not contained within the 95% confidence interval.
Results: 44 total proteins were identified between the three samples. Proteins known to be associated with the nuclear envelope (Lamin A/C and Myoferlin) and nuclear pore complex (Myosin-lc) as well as ten other proteins known to be associated with the nucleus (Annexin A1, Fructose-bisphosphonate aldolase A, Histone H2A type 1-C, Histone H2B type 1-B, Histone H3-like, Histone H4, Neuroblast differentiation-associated protein AHNAK, Polymerase I and transcript release factor, Prohibitin, and Protein kinase C delta-binding protein) were identified but were equally expressed between the cell types. Annexin A2, Annexin A4, CD44 antigen, and Histone H3-like proteins were found to be in statistically significant greater abundance in GF than in PDLF.
Conclusion: This study constitutes the first attempt in the literature to define periodontal proteomics. The quantitative and qualitative proteomic analysis demonstrated differences in two cytoplasmic and one extracellular fibroblast protein, but no differences were found within the nuclear envelope. Concerns of proper membrane-associated protein solubilization were raised based upon the paucity of known proteins identified.