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INTRODUCTION AND OBJECTIVE: With the advent of high-throughout technologies as a means to identify additional bladder cancer biomarkers and profiles of high diagnostic accuracy, we attempt at evaluating a two dimensional gel-based proteomic technology (2D-DIGE) for a more comprehensive exploration of the urinary proteome.

MATERIAL AND METHODS: Urine specimens belonging to individuals presenting microscopic hematuria under suspicion of bladder cancer were utilized for protein profiling. The presence of the disease was confirmed by the gold standard diagnostic method. Using 2D-DIGE analysis, protein extracts from control and bladder tumor samples were labelled with fluorescent dyes. Samples were run using IPG strips for the IEF (pH: 3-10, NL, 14x16 cm) and standard continuous SDS-PAGE for the second dimension. Proteins were captured and visualized using a fluorescence scanner (Typhoon 9400).Computational analyses of DIGE images were performed with the DeCyder software. Differentially expressed proteins were excised with the Ettan-Picker and further digested with trypsin. Identification or proteins was performed on a 4800 MALDI TOF/TOF analyzerin positive ion reflector mode.

RESULTS: 2D-DIGE gels identified over 2,000 identifiable spots differentially expressed between non-neoplastic urine samples (labeled with Cy3) versus urinary specimens belonging to patients with bladder cancer (labeled with Cy5). An internal pool generated by combining equal amounts of extracts from all the neoplastic and control urinary specimens was labelled with Cy2 dye, included in all gel runs, and served to assess interassay reproducibility. Among the spots detected, quantitative results indicated an increase of 9.9% proteins in neoplastic urinary specimens. Computational analyses of the images revealed changes in abundance of tumor vs control spots volume ratios within the 95th confidence level (Student’s t-test; p< 0.05). Interestingly, MALDI-TOF peptide mass fingerprinting identified 20 proteins differentially expressed between tumor and normal urinary specimens.

CONCLUSIONS: The 2D- DIGE proteomic strategy was able to identify urinary proteomic patterns indicative of the presence of bladder cancer. The current data strongly supports the value of 2D gel profiling of urine in combination with MALDI-TOF as a biomarker discovery approach for bladder cancer detection.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA