To discovery of new useful biomarkers for colon cancer, we performed Expression Difference Mapping analysis on 39 human cancer cell lines using SELDI ProteinChip platform. Several proteins were highly expressed in colon cancer cells (t-test, P<0.05), especially 12kDa protein showed the lowest p-value (0.0005) on SAX2. We then optimized the purification conditions for this candidate on chip, and directly transferred to conventional chromatography to purify. The purified protein was digested with trypsin or V8 protease to produce peptide mass fingerprinting analysis and collision-induced dissociation (CID)-tandem MS analysis with ProteinChip interface. Both identification results showed the candidate biomarker was a full-length of prothymosin-a. To evaluate whether prothymosin- a is a potential biomarker for colon cancer or not, we compared with the expression level of this protein between cancer cells and normal cells using real patients tissue samples. The peak intensity of identified prothymosin- a in colon cancer cells was clearly higher than that in normal colon cells in each patient. mRNA expression level of this protein in colon cancer was also higher than that in normal cells. Then we tried to capture prothymosin- a using antibody by Interaction Discovery Mapping analysis. The results of Interaction Discovery Mapping analysis was correlated with the results of Expression Difference Mapping analysis, it showed that the evaluation assay for this biomarker candidate could perform on SAX2 chip without establishment of antibody assay. These findings indicate that prothymosin- a could be a potential biomarker for colon cancer, and that the ProteinChip platform could rapidly perform the whole biomarker assay process from biomarker discovery stage to evaluation stage for the identified marker.
