Abstract
Purpose :
To date, little is known about the relationships of aqueous humor (AH) proteome with the mechanisms and severity of glaucoma. Here, we compared global AH proteome in early and advanced glaucoma patients and non-glaucomatous cataract patients.
Methods :
Forty glaucoma patients [n=20 each for early, Humphrey visual field (HVF) 24-2 mean deviation (MD) between -6 & -12 dB and advanced glaucoma, MD<-20 dB] aged >50 years undergoing glaucoma surgery were included. 30 age-matched cataract patients were included as non-glaucomatous controls. 0.8mL AH was harvested before surgery and stored at -80°C till use. Total protein concentration was measured by BCA assay and proteomic analysis done through global profiling with label free quantification (LFQ), data-independent acquisition (DIA) and ELISA. Quantitative global profiling was done by LC–MS/MS using the Q Exactive orbitrap plus hybrid mass spectrometer. All LFQ, DIA and ELISA intensities were processed using a log2 scale and proteins with expression greater than ±1.5 fold change were considered as differentially expressed proteins (DEPs). Database search and quantitative analysis was executed by subjecting the spectra to MaxQuant processing against Uniprot human database.
Results :
Between-group ANOVAs were significant for 171 of 494 proteins identified with LFQ (All p’s <0.001) (Fig. 1A). Bonferroni-corrected independent t-tests comparing early and advanced glaucoma identified 21 downregulated and 32 upregulated proteins as glaucoma progressed to a more severe stage (Fig.1B). Top 40 DEPs identified by LFQ in glaucoma were validated with DIA (Fig 1C). Further confirmation with ELISA showed a total of 14 DEPs including AQP4, CHT1, FGFR1, IDH3A, IGFBP6, IL2, IL4, MAPK15, mTORC1, mTORC2, SUCLG2, TGFB2, TIGR, and TNFa (Fig 1D) with roles to play in inflammation, insulin signaling, acetylcholine metabolism, and mitochondrial function. ELISA also showed significant differences in the expression of 9 DEPs with increases in AQP4, CHT1, IGFBP6, and IL2, and decreases in FGFR1, IDH3, IL4, SUCLG2, and TNFa as the disease progressed (Fig 1E).
Conclusions :
Progression of glaucoma may involve changes in the AH proteome mediated through inflammation, insulin signaling, and mitochondrial dysfunction.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.