Differential Proteomics Analysis Of Cytoplasmic And Membrane Proteomics Between Immortalized Cervical Cells And Cervical Cancer Cell

Ojective: To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from immortalized human endocervical cell (H8) and cervical cancer cell (Caski), and to identify the differential expressions of cytoplasmic and membrane proteins.Methods:1. H8 cells and Caski cells were incubated in RPMI 1640, the cytoplasmic and membrane proteins of them that are in their logarhytmic growth phase at approximately 80% confluence(3×106) were extracted by ProteoExtract Subcellular Proteome Extraction Kit.2. The cytoplasmic and membrane proteins of H8 cells and Caski cells were seperated by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). After being silver stained,The differential expression proteins were analyzed by using ImageMaster 2D analysis software.3. The selected protein spots were cut out of 2-DE gels for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis which was under a reflector mode, and then identified with peptide mass fingerprint (PMF) by searching in Mascot Science.Results 1. We obtained well-resolved,reproducible 2-DE patterns of the cytoplasmic and membrane proteins of H8 cells and Caski cells by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE) and ProteoExtract Subcellular Proteome Extraction Kit.2. 2-DE profiles show that were located mostly in the range of mass 10-180kDa and isoelectric points(pI)pH4-8. Approximately 1123 protein spots were resolved and detected by cytoplasmic of H8 cells and 1231 protein spots were detected by cytoplasmic of Caski cells in 2-D gel maps from pH3-10L IEF and the correlation coefficient between treated sample and control sample was about 70%. In addition, 8 notable differential protein spots were defined between them. Moreover, 2-DE profiles show that these membrane proteins in H8 cells and Caski cells were located mostly in the range of mass 10-200kDa and isoelectric points(pI)pH4-9. Approximately 1107 protein spots were resolved and detected by cytoplasmic of H8 cells and 1033 protein spots were detected by cytoplasmic of Caski cells in 2-D gel maps from pH3-10L IEF and the correlation coefficient between treated sample and control sample was about 71%. 13 notable differential protein spots were defined between them.3. 8 differentially expressed proteins spots between these cytoplasmic proteins in H8 cells and Caski cells were further identified by MALDI-TOF-MS analysis and 4 of them were successfully identified and characterized by PMF database searching. Among which, S100P,Ikkb(Inhibitor of nuclear factor kappa-B kinase subunit beta)were not identified in cytoplasmic proteins in Caski cells, CDK6 (Cell division protein kinase 6,TAF2(Transcription initiation factor TFIID subunit 2),STK3(Serine/threonine-protein kinase 3),TRAF5(TNF receptor-associated factor 5) were ignificantly up-regulated in cytoplasmic proteins in Caski cells.4. 13 differentially expressed proteins spots between these membrane proteins in H8 cells and Caski cells were further identified by MALDI-TOF-MS analysis and 3 of them were successfully identified and characterized by PMF database searching, LRC8D(Leucine-rich repeat-containing protein 8D),EDAR(Tumor necrosis factor receptor superfamily member EDAR precursor),Metaxin-1 were significantly up-regulated in membrane proteins in H8 cells.Conclusions: 6 differentially expressed cytoplasmic proteins spots proteins and 3 differentially expressed membrane proteins spots proteins between H8 and Caski cells were defected by means of immobilized pH gradient-based two-dimensional gel electrophoresis (2-DE). The differential expression proteins of nucleus may be valuable for studying mechanisms of cervical carcinogenesis, or as diagnostic markers and therapeutic targets for cervical cancer.