Protein-residual ECM N-glycosylation delineates a composition-resolved myCAF-enriched stromal state in pancreatic ductal adenocarcinoma

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is embedded in a dense stromal microenvironment, but total extracellular matrix abundance alone does not define its molecular state. We tested whether extracellular matrix (ECM) N-glycosylation provides a post-translational readout of CAF-associated stromal biology beyond matched protein abundance. Methods We integrated peptide-level and site-level CPTAC-PDAC N-glycoproteomics from 135 PDAC tumors with matched proteomics, RNA expression, clinical covariates, external transcriptomic cohorts, and spatial transcriptomics; five adenosquamous tumors were retained for sensitivity analysis. ECM glycopeptide and glycosite features were residualized against matched protein abundance to generate protein-residual ECM N-glycosylation scores. Robustness analyses evaluated nonlinear residualization, detection-probability weighting, feature coverage, acquisition plex, and feature resampling, with additional analyses of CAF subtypes, glycan composition, spatial localization, and clinical context. Results Residual ECM N-glycoprotein scores were associated with conventional myCAF (adjusted beta = 0.426 and 0.425) and CAF/stroma phenotypes (beta = 0.304 and 0.312) after adjustment for proteome matrisome score, stromal fraction, and neoplastic cellularity. The signal was distributed across a candidate ECM glycoprotein program and remained stable under nonlinear residualization, detection-probability weighting, observation thresholds from 40 to 110 tumors, and random feature splitting; the corresponding candidate-gene RNA program localized to CAF-rich spatial niches. Composition-defined high-mannose glycoforms showed the strongest myCAF effects (beta = 0.552 and 0.549), and the candidate program was higher in GeoMx CAF than epithelial segments in all 20 patients (mean paired difference = 1.234, 95% bootstrap CI 1.054–1.389; p = 9.57 × 10 −5 ). Conclusions Protein-residual ECM N-glycosylation identifies a measurable, composition-resolved myCAF-associated stromal layer in PDAC and nominates high-mannose ECM glycoforms and recurrent ECM glycoproteins as potential translational candidates for glycosite-resolved experimental validation.