Relative Quantification of Protein Expression in Metastatic Clear Cell Renal Cell Carcinoma Tissue Using iTRAQ LC-MS/MS Analysis
Nicole MA White, Olena Masui, Leroi DeSouza, Olga Krakovska, Ajay Matta, KW Michael Siu, George M Yousef. St. Michael's Hospital, Toronto, ON, Canada; York University, Toronto, ON, Canada
Background: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The five-year survival rate for metastatic RCC is <10%. There are currently no biomarkers in use for clinical diagnosis or prognosis of clear cell renal cell carcinoma (ccRCC).
Design: We performed iTRAQ (isobaric tags for relative and absolute quantitation) analysis on six pairs of primary ccRCC and normal matched kidney from the same patient in addition to six metastatic RCC samples. Samples were homogenized, clarified, trypsinized, labeled, and pooled. The pooled labeled digests were then separated by offline strong cation exchange chromatography, followed by on-line reverse phase nano-liquid chromatography mass spectrometry (LC-MS/MS) analyses in triplicate. An exclusion list was used to minimize redundancy in subsequent iterations and consisted of the mass-to-charge (m/z) ratios and elution times of peptides that had been identified in the previous iteration(s). MS data were analyzed by ProteinPilot.
Results: Using a cut-off of 5% local false discovery rate, a total of 1,256 non-redundant proteins were identified in a total of five sets, from which 456 proteins were confidently quantified (p≤0.05). 39 proteins were differentially expressed in the majority of cancer (primary and metastatic) samples versus the non-malignant pool, while another 20 were differentially expressed in the majority of metastatic versus primary samples. Three overexpressed proteins chosen as best candidates as potential ccRCC prognostic biomarkers are currently being independently verified by western blot and tissue microarray analysis. All differentially expressed proteins will be used as foci in a pathway analysis to elucidate their possible roles in tumorigenesis.
Conclusions: The identification of clinical markers for RCC patients will have a great impact on patient care since it will significantly reduce the risk of complications of late stage disease and will lead to significant reduction of the cost of treating advanced metastatic disease. Also, the mechanism of RCC metastasis remains to be elucidated. Bioinformatic analysis of dysregulated proteins in metastatic RCC will shed light on the pathways that contribute to this malignancy leading to a further understanding of RCC pathogenesis and forming the foundation for the design of new targeted therapies.
Category: Genitourinary (including renal tumors)
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 92, Wednesday Morning