Identification of very early stage disease, including cancer in situ, is currently the most promising approach to reduce pancreatic ductal adenocarcinoma (PDAC) mortality. Development of serum biomarkers for early diagnosis is particularly significant for tumors located in the pancreas, an organ that is relatively inaccessible to examination. The search for clinically useful biomarkers in PDAC has been challenging. The well-defined genetic pathway from normal ductal epithelium to invasive PDAC suggests that genetic alterations predate development of invasive and incurable cancer. As such, there is the potential to detect the resulting protein and PTM changes before the tumor has progressed to an incurable stage. Unfortunately, all biomarkers investigated to date lack the sensitivity and specificity to serve as a clinically useful screening test. This proposal therefore addresses a major unmet clinical need: currently, no strategy has been developed to effectively detect early stage PDAC. We have recently developed two complementary approaches to identify potential biomarkers. The first seeks to identify and quantify PDAC-derived protein biomarkers in serum using an isotopic mass-labeled proteome, produced and/or secreted by cultured PDAC cells, as an internal standard. The second approach quantifies activities of selected proteases, a class of enzymes that plays an important role in cancer biology. We propose to expand upon the serum protein and enzymatic biomarkers that we have discovered, by improving the analytical sensitivity and by analyzing carefully collected, statistically relevant and gender- and age-matched sample sets (PDAC; pre-cancerous; chronic pancreatitis; etc.). Central to our efforts will be (1) mass spectrometry-based protein quantitation and fluorescence-based protease activity assays, (2) deep and quantitative proteome and phosphoproteome analysis of cancer cells, secretomes and minute amounts of PDAC tumor tissues, and (3) tumor cell type-specific cellular and secreted protein reference standards, termed Super-SILAC and SEC-super-SILAC, that enable accurate relative quantitation of selected proteins in biological fluids and tumor tissues. We propose to test these hypotheses under the following three highly integrated study aims: (1) We will characterize panels of cellular, secreted, and tumor tissue proteins specific for PDAC as well as panels of serum proteases and prioritize candidate biomarkers for further development based on four criteria met. (2) We will then study protein and protease biomarker candidates in human serum. We hypothesize a subset of these biomarker candidates are differentially expressed or active in serum from early PDAC versus benign pancreatic disease. (3) Biomarkers that show promise will be subjected to verification by using larger sample cohorts to build mixed (i.e., proteins and protease activities) multivariate PDAC prognostic models. Performance characteristics of this model will be determined and validated for clinical use. Development of effective, noninvasive biomarkers for screening patients at risk for PDAC, where none currently exist, will have a significant impact on the treatment and clinical course of this disease.