TMT-based quantitative proteomic analysis of vitreous humor in proliferative diabetic retinopathy

OBJECTIVE: To study the changes in vitreous humor protein profile of proliferative diabetic retinopathy (PDR) and macular hole (MH)/epimacular membrane (EMM), and to identify potential candidate biomarkers for PDR by screening differentially expressed proteins (DEPs). METHODS: This study used tandem mass tag (TMT) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify DEPs in vitreous samples from PDR patients and MH/EMM control patients. The identified DEPs were analyzed via bioinformatics methods to screen candidate proteins with research potential. Parallel reaction monitoring (PRM) technology was used to perform targeted validation of 30 proteins with potential regulatory effects in PDR. RESULTS: A total of 4812 peptides and 877 proteins were isolated and identified via TMT combined with LC-MS/MS proteomic analysis, including 59 DEPs (fold change > 1.2, P < 0.05). Bioinformatics analysis showed that DEPs were mainly involved in critical biological processes including cellular process, stimulus-response, biological regulation, localization, immune system process and metabolic process. A total of 23 proteins were successfully quantified via PRM validation, and the overall expression trends were consistent between TMT-based quantification and PRM results. Among them, coagulation factor V (FV), profilin-1 (PFN1), thioredoxin (TXN), ferritin light chain (FTL) and selenoprotein P (SeP) not only showed consistent and significant quantitative trends, but also reached statistical significance (P < 0.05) in both discovery and validation cohorts. CONCLUSION: This study revealed significant alterations in the vitreous humor protein profile of PDR patients. The five identified candidate proteins (FV, PFN1, TXN, FTL, SeP) may serve as potential candidate biomarkers for PDR, providing new research directions and therapeutic targets for exploring the pathogenesis of PDR.