1517 Differential Extraction of the Gingival Fibroblast Nuclear Membrane Proteome

Saturday, March 24, 2012: 9:45 a.m. - 11 a.m.
Presentation Type: Poster Session
H. MCKNIGHT, Periodontology, Ohio State University, Columbus, OH, T. HART, Department of Periodontics (MC 859), University of Illinois at Chicago, Chicago, IL, D.N. TATAKIS, Periodontology - College of Dentistry, Ohio State University, Columbus, OH, and A. MARIOTTI, Dentistry, Ohio State University, Columbus, OH
Objectives:  The composition of nuclear membrane proteins (NMP) of fibroblasts derived from the periodontium is unknown.  Identification of NMP is complicated since extraction of membrane proteins can be difficult due to the lipophilic nature of these molecules.  The purpose of this study was to compare the quantity of unique identifiable membrane proteins using a single enzymatic wash preparation versus a two wash preparation for digestion and denaturation in proteomic analysis.

Methods:  GF used were obtained from 2 human biopsies derived from non-inflamed tissue in the retromolar pad area.  Cells were cultured and propogated in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum.  GF were harvested when confluent in the 4th cell culture passage.  NMP were isolated by differential centrifugation.  15 μg of protein from each sample was digested with trypsin or trypsin followed by a urea wash, followed by iTRAQ™ mass labeling for peptide qualitative and quantitative identification using liquid chromatography and mass spectrometry (LC-MS/MS) proteomic analysis methods. 

Results: 748 unique proteins were identified when a trypsin wash was used for digestion and identification of GF NMP with LC-MS/MS analysis.  25 of these proteins were known to be nuclear membrane or associated proteins.  When a 2M urea wash was performed, 573 proteins were identified; 19 of which are nuclear membrane or associated proteins.  In total 31 unique nuclear membrane or associated proteins were identified by proteomic analysis.

Conclusions: Proteins identifiable by proteomic analysis were dependent on the processing enzymes and washes.  The addition of a urea wash for protease digestion and denaturation for LC-MC/MC directly affected the proteins identified.  These results are essential to gaining information on the protein components of GF and for further exploration into cell function.  

Keywords: Cell biology, Fibroblasts, Periodontium-gingiva and Proteins