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Pancreatic cancer is among the most lethal malignancies worldwide. We aimed to identify novel prognostic markers by applying mass spectrometry (MS)-based proteomic analysis to formalin-fixed paraffin-embedded (FFPE) tissues. Resectable, node positive pancreatic ductal adenocarcinoma (PDAC) with poor (n = 4) and better (n = 4) outcomes, based on survival duration, with essentially the same clinicopathological backgrounds, and noncancerous pancreatic ducts (n = 5) were analyzed. Cancerous and noncancerous cells collected from FFPE tissue sections by laser microdissection (LMD) were processed for liquid chromatography (LC)-tandem MS (MS/MS). Candidate proteins were identified by semiquantitative comparison and then analyzed quantitatively using selected reaction monitoring (SRM)-based MS. To confirm the associations between candidate proteins and outcomes, we immunohistochemically analyzed a cohort of 87 cases. In result, totally 1,229 proteins were identified and 170 were selected as candidate proteins for SRM-based targeted proteomics. Fourteen proteins overexpressed in cancerous as compared to noncancerous tissue showed different expressions in the poor and better outcome groups. Among these proteins, we found that three novel proteins ECH1, OLFM4 and STML2 were overexpressed in poor group than in better group, and that one known protein GTR1 was expressed reciprocally. Kaplan–Meier analysis showed high expressions of all four proteins to correlate with significantly worse overall survival (p < 0.05). In conclusion, we identified four proteins as candidates of prognostic marker of PDAC. The combination of shotgun proteomics verified by SRM and validated by immunohistochemistry resulted in the prognostic marker discovery that will contribute the understanding of PDAC biology and therapeutic development.While the search for biomarkers for particular cancers has often focused on mRNA, protein profiles may actually be more accurate. In addition, mRNA levels can't detect the activation of key signaling molecules in protein networks. In this study of pancreatic cancer, the authors used a novel strategy combining “global shotgun proteomics” using mass spectrometry (MS), and targeted “selected reaction monitoring” (SRM). They found that patients whose tumors expressed the proteins ECH1, OLFM4, STML2 and GTR1 had significantly worse outcomes. These proteins may thus have prognostic significance, and may also suggest new therapeutic targets.