Biomarker Discovery of Colorectal Cancer Using Membrane Proteins Extracted from Cancer Tissues

Colorectal cancer is the leading cause of cancer-related death in women and third leading cause of death in men. The prognosis worsens when cancer metastasizes to other organs. Thus, there is an urgent need to develop biomarkers for the early diagnosis of metastasis as well as cancer development. Mass spectrometry-based technologies have been applied to the discovery of protein biomarkers, especially in the field of cancer. These results have identified numerous candidate protein biomarkers. Unfortunately, only a few are currently being applied in clinical diagnostics. Recent advances in proteomic technology such as selected/multiple reaction monitoring (SRM/MRM) facilitated the detection and quantitation of specific proteins in complex samples without the need for antibodies. We performed a quantitative proteomic analysis of membrane proteins extracted from colorectal cancer tissues using the iTRAQ shotgun method to discover biomarker candidates, and then extensively validated the biomarker candidate proteins by SRM/MRM. A total of 5,566 proteins were identified in tissue samples obtained from adenoma and cancer with and without metastasis. Differences were observed in the expression of about 400 proteins. Among them, 105 biomarker candidates which were predicted to be membrane proteins and extracellular proteins by gene ontology analysis were quantitated using SRM/MRM. As a result, we could confirm differences in the expressions of 69 of these proteins using the same set of patient samples as discovery experiments, and this was subsequently verified in an independent set of samples. Significant differences were observed in the expression of 44 of these proteins. Moreover, some of the biomarker candidates were detected and quantitated in the serum of colorectal cancer patients. These biomarker candidates are promising diagnostic tools on investigating the development and progression of colorectal cancer.