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To search for potential protein markers of colorectal cancer (CRC), the changes in protein expression levels between microdissected tumor cells and normal mucosa epithelia were analyzed by an acetylation stable isotopic labeling method coupled with linear quadrupole ion trap fourier transform mass spectrometry (LTQ-FTMS). In total, 137 proteins were up-regulated or down-regulated significantly in cancer by at least two-fold. Based on gene ontology analysis, the largest part of differential proteins were unknown for both subcellular localization and biological process. In particular, the significant up-regulation of transgelin-2 (TAGLN2) in CRC was validated by Western blot analysis and further evaluated by immunohistochemistry in paired tumor and normal mucosa samples from 120 consecutive CRC patients, 20 adenomas, and eight synchronous hepatic metastases of CRC. TAGLN2 expression was frequently observed in cancer cells, precancerous lesions, and hepatic metastases, whereas in normal epithelia expression was rarely observed. The overexpression of TAGLN2 was associated with lymph node and distant metastasis, advanced clinical stage (P < 0.001), and shorter overall survival in CRCs. Cox regression analysis indicated that high tumor-TAGLN2 expression represents an independent prognostic factor. Consequently, over-expression of TAGLN2 may serve as a new biomarker for predicting progression and prognosis of CRC. (Cancer Sci 2009)