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Seminar 14: Bile Proteomics for Marker Discovery in Cholangiocarcinoma

“Bile Proteomics for Marker Discovery in Cholangiocarcinoma”
by: Dr. Glenn Kunnath Bonney MbChB, MRCS, MD
Department of Liver Transplantation
St James University Hospital
Leeds, United Kingdom
DATE: 22 November 2013 (Friday)
TIME: 3 – 4 p.m
VENUE: Lecture Room 4, Applied Sciences Building, QIUP


Cholangiocarcinoma is a cancer of the biliary epithelia with rising incidences and 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 downstream 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 (CCA), 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 but has not as yet been described in bile. Using a protocol developed in-house, we carried out an analysis of bile from patients with CCA and compared this with 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 up-regulation in CCA. This latter spot was identified as Glutathione-S-Transferase pi by MS/MS analysis and differential expression was subsequently confirmed in validation studies.The work carried out has provided a reproducible method of processing bile for proteomic analysis. With the technique, we have successfully carried out two parallel proteomic-based approaches resulting in the discovery of three biliary proteins that show promising early results as potential biomarkers for CCA. Further validation studies and newer techniques in this area are currently being undertaken.

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