Biomarkers for colorectal cancer (CRC) early diagnosis are currently lacking. The purpose of this study was to interpret molecular events in the early stage of CRC that may bring about new biomarkers for early diagnosis. Methylation isotope labeling assistant gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS) strategy was developed to improve protein identification in quantitative proteome analysis between pooled early stage CRC and pooled normal counterparts. Expression of candidate biomarkers were in situ verified in a 372-dots tissue array, and their relative concentrations in sera were validated in 84 CRC patients and healthy individuals. Altogether, 501 proteins showing consistent differential expression were discovered. Function analysis highlighted the ubiquitination-proteasome and glycolysis/gluconeogenesis pathways as the most regulated pathways in CRC. Two glycol-proteins, alpha1 antitrypsin (A1AT) and cathepsin D (CTSD), which play central role in proteasome regulation, were further examined due to their possible importance in human cancers. Consistent with proteome data, CRC specimens expressed less A1AT and more CTSD than normal counterparts in both tissue and serum levels. By combining CTSD and A1AT, 96.77% of CRC tissues were distinguished from normal tissues by immunohistochemical analysis on a tissue array (P<0.0001). Combined CTSD and A1AT should be strongly considered for clinical use in early diagnosis of early stage CRC, and the methylation assistant GeLC-MS approach is competent for a global quantitative proteome study.