Proteomic analysis of FFPE tissue samples is important for the detection of proteins that are involved in tumor progression, with the potential to find new disease-specific biomarker to improve diagnostics. For pancreatobiliary (PB) cancer a specific biomarker for diagnostics is not yet known, except the tumor markers CA 19‑9 and CEA, which are used for follow‑up purposes but cannot be used for early diagnosis of PB cancer due to their elevation also in benign conditions, such as pancreatitis. The early stage in tumor progression is associated with a transition of normal cells into so-called precursor lesions, which seem to develop along similar pathways and show numerous morphological and molecular similarities. The group of Prof. Esposito at the Institute of Pathology developed methods for morphomolecular characterization of PB precursors as well as molecular subtyping of different precursor stages applied on formalin-fixed, paraffin-embedded (FFPE) tissues. For the characterization of PB cancer, several proteome studies have been attempted to detect proteins involved in progression. However, they provided only limited insight into the tumorigenic mechanism of PB cancer, because the results were biased by signals from different cell types such as acinar, endocrine, stromal and inflammatory cells. In this study we optimized and applied sensitive methods combining microdissection and quantitative, label‑free proteomics to gain a more comprehensive understanding in the development of PB cancer. By using a data independent acquisition (DIA) mode for LC-MS/MS analysis the goal is a combined proteomic and glycoproteomic (specific O‑glycans) analysis to unravel differences in malignant and benignant tissue transformation of pancreatic precursor lesions. These findings can support clinical decisions and improves early cancer diagnostics as well as give insights to regulatory processes provided by glycosylation patterns. Pathway analysis reveals enrichment of transport processes from ER to Golgi, RNA splicing and glycolytic processes in malignant progression. We observed one protein candidate, arising from differential analysis of the proteomic dataset that is strongly associated with ER activity. Herein we confirm RNA expression data from literature and give contradictory results to the current biomarker validation via antibody staining. Within the Glyco‑DIA approach we found overall enrichment of sialylated glycoproteins, as well as glycoproteins carrying the core‑1 glycan in malignant progression. Differences in the glyco‑modification type is observed for 14‑3‑3 theta, in which the peptide carrying the core‑1 glycan (T antigen) is enriched in malignant progression. For this modification, literature evidence is not yet given and the results need further validation by external methods.