B10
The pancreatic cancer serum proteome: highly abundant proteins and associated biomarkers
Mark Aspinall-O'Dea1, Sarah Tonack1, Rosalind Jenkins2, Victoria Elliott2, Neil Kitteringham2, John Neoptolemos1, Eithne Costello1
1University of Liverpool, Division of Surgery and Oncology, Liverpool, Merseyside, UK, 2University of Liverpool, Department of Pharmacology and Therapeutics, Liverpool, Merseyside, UK
Background
Immunoaffinity columns which remove abundant serum proteins generate depleted (DF) and corresponding bound (BF) protein fractions resulting in proteome simplification. The BF contains elements such as albumin which associate with lower abundance serum proteins. Here we describe the quantitative analysis of BF from pancreatic cancer patients and controls using iTRAQ and two-dimensional liquid chromatography (2DLC) coupled tandem mass spectroscopy (MS/MS).
Method
Four sample groups (Pancreatic Cancer, Chronic Pancreatitis, Bile Duct Obstruction and Healthy Control; 15 individuals per group, 50μl per individual) were collected and pooled following quality control using silver-stained 4-15% SDS-PAGE. Serum delipidation (modified from Cham and Knowles 1976) was undertaken prior to depletion with a Proteoprep® 20 Plasma Immunodepletion Kit (Sigma-Aldrich). The resultant BF was collected and buffer exchanged into 0.5M Sodium Carbonate solution using centrifugal bioseparation (4,500 X g 20 min, Microsep 30K, Pall) and concentrated toto ~1mg/ml.. Samples were iTRAQ labelled (Applied Biosystems) using either a 4-plex or an 8-plex iTRAQ kit. Samples fractionated by 2DLC coupled to automated, rapid sample injection (NanoMate 100, Advion Biosciences) on to a Q-Star Mass Spectrometer.
Results
Thirty-seven proteins, with quantification data between the cancer pool and controls, were identified in total. Selective albumin removal is being undertaken to further improve the number of proteins identified.
Conclusion
A number of these proteins, such as Ceruloplasmin and Apolipoprotein A1 are associated with other cancers; however validation of candidates which display the strongest association with our cancer pool is being undertaken.
