Purpose: To analyze protein patterns in the aqueous humor of glaucoma patients in comparison to control subject using two different methods.
Methods: Aqueous humor was collected from 52 patients with primary open-angle glaucoma (POAG) and from 55 control subjects (CO). Twenty-two POAG samples and 24 CO samples were used for protein profiling through surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. The data were analyzed by multivariate statistical methods and artificial neural networks. One highly significant biomarker was identified through matrix assisted laser desorption/ionisation time of flight-mass spectrometry (MALDI-TOF). Thirty samples from patients with POAG and 31 control samples were analyzed through two-dimensional electrophoresis. Subsequently, the protein spots of all gels were detected, and the two groups were compared. One spot group exhibiting clear differential abundance was identified by mass spectrometry (electrospray ionization mass spectrometry).
Results: In the samples analyzed by SELDI-TOF-MS, about 250 protein peaks could be consistently clustered in both groups. The analyses revealed eight biomarkers, which discriminated glaucoma from non-glaucoma controls with a sensitivity of 90% and a specificity of 87%. These biomarkers were purified further, and one marker, which was upregulated in glaucoma patients (p=0.006), was identified as transthyretin. The upregulation of transthyretin in POAG patients was also confirmed by enzyme linked immunosorbent assay (ELISA; p=0.03). In all samples analyzed by two-dimensional electrophoresis, complex protein patterns were detected in a total of 177 spot groups. The aqueous humor of all glaucoma patients revealed some regions that were clearly different from the controls. Several spots were significantly increased in the aqueous humor of glaucoma patients. One of the proteins that is highly abundant in the aqueous of glaucoma patients was identified as transthyretin.
Conclusions: The aqueous humor of glaucoma patients revealed characteristic differences in protein/peptide profiles from control patients using two different analytical methods, SELDI-TOF-MS and two-dimensional electrophoresis. Interestingly, we could detect elevated transthyretin concentrations in glaucoma samples. Transthyretin might play a role in the onset of glaucoma since it has been shown to form amyloid deposits. These particles could cause outflow obstructions thereby increasing intraocular pressure as a possible onset mechanism.