Abstract Introduction: The quantitative evaluation of expression and post-translational modification of receptor tyrosine kinase targets in tumor tissues may enable the development of a molecular profile for the selection of targeted therapy in colon cancer. Experimental Procedures: Colon cancer cell lines are used to develop peptide-based quantitative assays for receptor tyrosine kinase protein expression and post-translational modification using SDS-PAGE protein fractionation prior to in-gel digestions and liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM). Additional downstream signaling proteins are also investigated. The sensitivity of each assay is tested using serial dilutions of cell lysate before translation to individual frozen tissue sections, which are bookmatched to hematoxylin and eosin stained slides for histology. A classification scheme for selection of targeted therapy is devised based on the amounts of protein expression and modification detected by LC-MRM. Data Summary: LC-MRM assays have been previously developed to monitor components of the beta catenin signaling pathway have been translated to measure protein expression in individual frozen tissue sections. Using the same methodology, LC-MRM assays have been developed for receptor tyrosine kinases (RTKs), including EGFR, Met, VEGFR, and IGF1R, as well as selected RTK interacting proteins and downstream signaling components. Implementing these novel assays in parallel with those already existing, an analysis of multiple signaling pathways in frozen section of colon tumors is conducted to enable a view of the tumor biology and assess the relevance of treatment with RTK inhibitors. Conclusions: The ability to examine cancer-related pathways in human tumors with quantitative mass spectrometry will produce improved understanding of the processes involved in tumor development and progression as well as elucidate the mechanisms of cancer cell response to chemotherapy. The coupling of quantitative proteomics with medical history and histology for comprehensive analysis of tumor tissues represents a promising step toward the ultimate goal of establishing a molecular basis for personalized medicine. This effort will demonstrate the feasibility of creating a quantitative proteomics method for tumor evaluation that could be used to translate basic science hypotheses into clinical samples and build a systems biology tool for patient assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5123. doi:10.1158/1538-7445.AM2011-5123