PMs-Ti-based plasma extracellular vesicles glycoproteomics and its application in lung cancer.

Extracellular vesicles (EVs) are densely decorated with glycoproteins and serve as pivotal molecular reservoirs in liquid biopsies. However, existing EV glycoproteomic analyses typically involve complex procedures such as EV enrichment, in-solution enzymatic digestion, glycopeptide enrichment, and N-glycosylation removal, making it difficult to obtain intact N-glycopeptide information. This study developed an analytical method based on Ti-functionalized porous microparticles (PMs-Ti) to achieve efficient analysis of intact EV glycopeptides: EVs were captured and digested in situ on PMs-Ti, followed by intact glycopeptide enrichment. Approximately 94% of the proteins identified by this method were core EV proteins, indicating a high degree of specificity. Compared with commercial EV isolation kit, the number of EV proteins identified in our experiments increased by approximately 6-fold, and the number of intact N-glycopeptides increased by about 1.4-fold, showing the method's significant advantages in plasma EV intact glycoproteomic analysis. Using the optimized workflow, quantitative analysis of plasma EVs from 7 lung cancer patients and 7 healthy controls identified 62 core differentially expressed proteins. Glycan analysis further revealed remodeling of EV glycosylation, characterized by increased sialylated-fucosylated glycans and decreased sialylated glycans. The results demonstrate that the proposed method provides a reliable platform for systematic profiling of EV glycosylation in cancer, facilitating the exploration of site-specific glycosylation patterns and their potential biological relevance.