Protein glycosylation profiling in lung adenocarcinoma and precursor lesions: analysis of FFPE tissue sections

Protein glycosylation is a major post-translational modification that regulates tumor initiation and progression; however, its dynamic modeling during multistep evolution of lung adenocarcinoma (LUAD) remains poorly understood, particularly in clinically archived tissues. Here, we established an integrated multi-omics workflow combining global proteomes, N-glycans, and site-specific intact N-glycopeptides to comprehensively characterize glycosylation in formalin-fixed paraffin-embedded (FFPE) specimens spanning four pathological stages of LUAD progression: inflammatory nodules (IN), atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), and invasive adenocarcinoma (IAC). Using optimized protein extraction, hydrophilic interaction liquid chromatography (HILIC)-based glycopeptide enrichment, and high-resolution LC-MS/MS, we achieved large-scale identification of proteins, N-glycans, and intact glycopeptides from archival clinical samples. Integrated analyses revealed progressive remodeling of site-specific N-glycosylation during malignant transformation, characterized by increased glycan branching, fucosylation, and sialylation during the transition from premalignant lesions to invasive cancer. Sialylated glycans reached their highest abundance in the premalignant AAH stage, whereas highly branched and fucosylated complex N-glycans predominated in invasive adenocarcinoma, indicating stage-dependent glycan remodeling throughout disease progression. Functional enrichment analyses linked these glycosylation alterations to extracellular matrix organization, neutrophil degranulation, and immune-associated pathways, while representative glycoproteins, including CEACAM6 and FGB, exhibited coordinated changes in protein abundance and site-specific glycoform micro-heterogeneity across pathological stages. Collectively, this study demonstrates the feasibility of deep glycoproteomic profiling using archived FFPE tissues and provides a comprehensive molecular atlas of glycosylation remodeling during LUAD progression. These findings establish a valuable resource for elucidating disease mechanisms and identifying stage-specific glycosylation biomarkers and potential glycan-targeted therapeutic candidates for early lung adenocarcinoma.