Identification of tumor antigens in renal cell carcinoma by serological proteome analysis

We have investigated the suitability of proteomics for identification of tu mor-associated antigens. First, we compared the proteomes of nontumorous ki dney and renal cell carcinoma (RCC) by two-dimensional get electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (simil ar to 800 spots in RCCs versus similar to 1400 spots in kidney). 2-DE immun oblotting revealed five RCC-specific spots, reproducibly reactive with RCC- patient but not healthy donor control sera. Two of these antigens were isol ated by preparative 2-DE, and identified by Edman sequencing of tryptic pep tides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is a n actin-binding protein of unknown function predominantly expressed in smoo th muscle cells. In situ hybridization revealed that SM22-alpha is not expr essed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usuall y not expressed in kidney. Interestingly, a different isoform (CAXII) has p reviously been identified by serological expression cloning as an antigen o verexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, res pectively, whereas sera from 13 healthy individuals did not react. In concl usion, serological proteome analysis may be a new tool for the identificati on of tumor-associated antigens.