Multi‐Site Proteomics Reveals Distinct Molecular Patterns Within Functional Dyspepsia

BACKGROUND: Functional dyspepsia (FD) is a common gastrointestinal disorder lacking established molecular targets. Symptom-based classification into epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS) often fails to guide effective therapy. OBJECTIVE: To identify molecular subtypes of FD using proteomics to better understand disease heterogeneity. METHODS: Using 4D-DIA proteomics, we analyzed gastric and duodenal biopsies from 47 FD patients and 10 healthy controls. Non-negative matrix factorization was applied separately to each site for unsupervised clustering. Subtypes were characterized by clinical profiles, pathway enrichment, immune infiltration analysis, and validated via histology and immunostaining. RESULTS: Two gastric (G1: 17%, G2: 83%) and three duodenal subtypes (D1: 13%, D2: 70%, D3: 17%) were identified. G1 showed strong immune activation linked to H. pylori infection, while G2 was enriched for metabolic pathways. Duodenal subtypes revealed distinct mechanisms: D1 featured eosinophil-predominant inflammation, D2 showed ROS metabolism and ferroptosis correlated with pain, and D3 exhibited mast cell infiltration. Gastric and duodenal subtypes were independent of each other. No significant association was found between molecular subtypes and EPS/PDS clinical classification. CONCLUSION: FD involves heterogeneous molecular mechanisms across gastric and duodenal sites. Duodenal profiling exhibited disease-relevant immune and oxidative pathways, while gastric patterns are largely distinguished by H. pylori infection status. Conventional symptom-based subtyping fails to capture this complexity, underscoring the need for mechanism-based stratification to guide personalized treatment in FD.