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PURPOSE: To investigate the aqueous humor proteome in patients with glaucoma and a control group. METHOD: Aqueous humor was obtained from five human donors diagnosed with primary open angle glaucoma (POAG) and five age- and sex-matched controls undergoing cataract surgery. Quantitative proteome analysis of the aqueous humor by hyper reaction monitoring mass spectrometry (HRM-MS) based on SWATH technology was performed. RESULTS: Expression levels of 87 proteins were found to be different between glaucomatous and control aqueous humor. Of the 87 proteins, 34 were significantly upregulated, whereas 53 proteins were downregulated in the aqueous humor from glaucoma patients compared to controls. Differentially expressed proteins were found to be involved in cholesterol-related, inflammatory, metabolic, antioxidant as well as proteolysis-related processes. CONCLUSION: Glaucoma leads to profound changes to the aqueous humor proteome consistent with an altered metabolic state, an inflammatory response and impaired antioxidant defense.