Abstract We prospectively collected matched tumor specimens, adjacent non-tumor tissues, and blood samples from 110 colon cancer patients and analyzed the samples using seven omics platforms, including whole-exome sequencing, copy number arrays, RNA-Seq, miRNA-Seq, label-free global proteomics, isobaric tandem mass tag (TMT) labeling-based global proteomics, and TMT-based phosphoproteomics. Comparative proteomic and phosphoproteomic analysis of paired tumor and adjacent normal samples produced the first comprehensive catalogue of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers and drug targets. These cancer-associated proteins and phosphosites had very little overlap with known cancer genes in the Cancer Gene Census, providing a novel information layer to our knowledge about colon cancer. One notable finding in differential proteome analysis is the identification of several cancer/testis antigens that were recurrently over-expressed in tumors compared to adjacent normal tissue, including IGF2BP3 (51%), SPAG1 (14%), and ATAD2 (8%). Through integrative analysis of the whole-exome sequencing, RNA-Seq, and proteomics data, we further predicted personalized neoantigens for 38% of the patients. In total, we found proteomics-supported neoantigens or cancer/testis antigens for 78% of the tumors in this cohort, demonstrating the potential of proteogenomics in identifying tumor antigens for cancer vaccine development. Proteomics data complemented somatic copy number analysis results and showed that multiple somatic copy number deletion events converge to repress the endocytosis pathway, suggesting its tumor suppressor role in colon cancer. In addition to reinforcing or complementing genomic findings, proteogenomic integration may also contradict genomics data-based inferences and lead to unexpected discoveries and therapeutic opportunities. Proteomics data identified SOX9 as an oncogene in colon cancer, whereas it was predicted to be a tumor suppressor based on somatic mutation data in the TCGA study. Phosphoproteomics data revealed a dual role of Rb phosphorylation in promoting proliferation and repressing apoptosis in colon cancer, clarifying the long-standing puzzle of colon cancer-specific amplification of this tumor suppressor and highlighting a unique opportunity for targeting Rb phosphorylation in colon cancer. Microsatellite instability status has been approved by the FDA as a biomarker for selecting patients for checkpoint inhibitor therapy in colorectal and other solid tumors. However, many MSI-high tumors fail to respond to checkpoint inhibition. Our proteogenomic analysis identified a subtype-specific association between increased glycolysis and decreased CD8 T cell infiltration in MSI-high colon tumors, suggesting glycolysis as a target for overcoming immune evasion in this MSI-H tumors. We make the primary and processed datasets available in publicly accessible data repositories and portals to allow broad use of these datasets for new biological discoveries and therapeutic hypothesis generation. Citation Format: Bing Zhang, Suhas Vasaikar, Chen Huang, Xiaojing Wang, Vladislav A. Petyuk, Sara R. Savage, Bo Wen, Yongchao Dou, Yun Zhang, Zhiao Shi, Osama A. Arshad, Marina A. Gritsenko, Lisa J. Zimmerman, Jason E. McDermott, Therese R. Clauss, Ronald J. Moore, Rui Zhao, Matthew E. Monroe, Yi-Ting Wang, Matthew C. Chambers, Robbert J. Slebos, Ken S. Lau, Qianxing Mo, Li Ding, Matthew Ellis, Mathangi Thiagarajan, Christopher R. Kinsinger, Henry Rodriguez, Richard D. Smith, Karin D. Rodland, Daniel C. Liebler, Tao Liu, CPTAC Investigators. Proteogenomic characterization of human colon cancer reveals new therapeutic opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-006.