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Title: A proteomic based approach for the identification of biliary markers of Cholangiocarcinoma
Author: Bonney, Glenn Kunnath
ISNI:       0000 0004 2740 3010
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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Cholangiocarcinoma is a cancer of the biliary epithelia with a rising incidence and a dismal prognosis. The lack of clinically useful biomarkers of the disease has led to the use of bile as a more proximal fluid in proteomic based biomarker discovery experiments. Early results have shown inherent difficulties in the acquisition, processing and reproducibility of this approach. By first establishing sample banking and developing a robust, streamlined method for processing bile for down stream proteomic investigations, we were able to perform two separate proteomic based analyses. Firstly, with the initial aim of cataloguing proteins in .bile from a patient with cholangiocarcinoma, we used a shotgun GeLC-MS/MS approach. By this method, .895 proteins were identified of which 621 had two or more significant peptides. A number of these proteins were of interest based on their known involvement in other cancers. Downstream studies on two of these molecules, Matrix Metalloproteinase-9 and Lipocalin-2, revealed up-regulation in CCA when compared to normal, benign and predisposed disease categories, suggesting they represent potential biomarkers. Secondly, we employed Difference Gel Electrophoresis (DIGE) in a comparative analysis to identify biliary markers of CCA. DIGE has greatly advanced the sensitivity and reproducibility of 2D PAGE based experiments and has, as yet, not been described in bile. Using a protocol developed in-house, we carried out a comparative analysis of bile from patients with cholangiocarcinoma and compared this between normal, benign and predisposed disease categories. By this method, 6 spots on 2D DIGE gels showed significant expression differences between disease categories with 5 showing down regulation and 1 showing upregulation in cholangiocarcinoma. This spot was identified iii as Glutathione-S-Transferase pi by MS/MS analysis and differential expression was subsequently confirmed in validation studies. The work carried out here has developed a reproducible method of processing bile for proteomic analysis and using this technique carried out two parallel proteomic based approach which has resulted in three biliary proteins that have shown promising early results. Further validation studies and newer techniques in this area are currently being undertaken.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available