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Abstract
Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers. Thus, here we provide a comprehensive protein expression map for clear cell renal cell carcinoma established by systematic comparative two-dimensional gel electrophoresis-based protein expression profiling of 16 paired tissue systems comprising clear cell renal cell carcinoma lesions and corresponding tumor-adjacent renal epithelium using overlapping narrow pH gradients. This approach led to the mapping of 348 distinct spots corresponding to 248 different protein identities. By implementing restriction criteria concerning their detection frequency and overall regulation mode, 28 up- and 56 down-regulated single target spots were considered as potential candidate biomarkers. Based on their gene ontology information, these differentially expressed proteins were classified into distinct functional groups and according to their cellular distribution. Moreover, three representative members of this group, namely calbindin, gelsolin, and heart fatty acid-binding protein, were selected, and their expression pattern was analyzed by immunohistochemistry using tissue microarrays. Thus, this pilot study provides a significant update of the current renal cell carcinoma map and defines a number of differentially expressed proteins, but both their potential as candidate biomarkers and clinical relevance has to be further explored in tissues and for body fluids like serum and urine.
Highlights
Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers
Systematic Comparison of Protein Expression Profiles of ccRCC Lesions and Corresponding Tumor-adjacent Kidney Epithelium—To determine the differential protein expression pattern of renal cell carcinoma (RCC), total protein extracts obtained from 16 tissue systems consisting of primary RCC lesions of the ccRCC subtype and corresponding tumor-adjacent kidney epithelium were subjected to classical 2DE using overlapping pH gradients of pH 4 –7 and pH 6 –11
Proteomics profiling is widely accepted as a tool for gaining insights into the dynamics of protein expression profiles reflecting complex cellular processes, including malignant transformation
Summary
Patients and Tissue Samples—Tissue samples from 16 primary ccRCC lesions and autologous tumor-adjacent renal tissue were obtained from RCC patients who had undergone radical nephrectomy at the University Hospital of the Johannes Gutenberg University in Mainz, Germany. Spots displaying a protein expression pattern restricted to either the tumor or the corresponding tumor-adjacent renal epithelium were selected for mass spectrometric identification. The heterogeneous data sets of the identified proteins were linked to the most recent Swiss-Prot identities (UniProt Knowledgebase Release 14.9), which served as the major source for additional information such as the chromosomal localization, and gene ontology, which provides information about gene function and cellular localization. Based on this information, the protein families and compartment localizations were assigned to each protein. The extent of immunostaining was scored according to the following criteria: negative, Ͻ5% positive cells; weak positive, 5–25% positive tumor cells; intermediate positive, 26 –50% positive tumor cells; and strong positive, Ͼ50% positive tumor cells
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