Transthyretin and complex protein pattern in aqueous humor of patients with primary open-angle glaucoma

Abstract

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.

Figure 1

Example of a two-dimensional gel with Oakley silver staining of aqueous humor sample from a POAG patient. The x-axis shows the first dimension (separation by isoelectric point), and the y-axis shows the second dimension (separation by molecular weight). The dark spots represent stained proteins.

Figure 2

Gel-like views of SELDI-TOF spectra of aqueous humor samples generated on two different chromatographic surfaces. The protein profiles on CM10 (weak cation exchange surface) chips can be seen on the left side and the protein profiles on H50 (reversed phase chemistry) chips on the right side. The profiles on the top are from control subjects and on the bottom from patients with POAG.

Figure 3

Group average intensities of mass spectrometry measurements for both CO and POAG groups and different laser energies. The intensities of the SELDI-TOF readings (U) were plotted against the molecular weight (in Da). All measurements on CM10 chips are shown in this graph. H: high, L: low energy. In several regions on this graph a higher protein intensity can be seen in the POAG group.

Figure 4

This scatter-plot demonstrates the correlation of the intensities of protein patterns between patients with primary open-angle glaucoma (POAG) and controls (CO). Spots above the line represent protein peaks with higher intensities in the POAG group (using SELDI-TOF analysis), dots below the line are peaks with lower intensity in the POAG group. This graph shows that up- and downregulated proteins were detected in aqueous humor of POAG patients.

Figure 5

The receiver operating characteristic curve for the diagnosis of glaucoma through complex protein patterns. The groups were discriminated with a sensitivity of 90% and a specificity of 87%. The calculation of the area under the curve (AUC) led to an r value of 0.94. The x-axis shows 1-specificity and the y-axis the sensitivity.

Figure 6

This graph shows the localization of spot groups 1-8 in a “master-gel” on the left side and the mean intensities of the control (CO) and primary open-angle glaucoma (POAG) groups on the right side. The spot groups 4, 7, and 8 existed in all samples and carried a higher amount of protein than all other spot groups especially in the POAG group. The only higher mean volume in controls can be seen in spot group 5.

Figure 7

Comparison of spot groups 1 and 2 (molecular weight 37.4–49.4 kDa, IP: 5.7–5.9). A shows a synthetic gel of all aqueous humor control samples (CO), and C shows a synthetic gel of all aqueous samples from patients with primary open-angle glaucoma (POAG). B shows a “real” 2D gel of an aqueous humor sample from a patient with primary open-angle glaucoma. The circle points out spot groups 1 and 2 on this gel. Different staining intensities due to the difference in protein concentration can be seen for spot group 1 and 2 on the two artificial gels.

Figure 8

Box plot of the protein concentration of the aqueous humor samples (mean±SE) of the control group and the primary open-angle glaucoma group at 14,132 Da from SELDI-TOF analysis. This protein was significantly upregulated in the POAG group (p=0.006) and was identified as human transthyretin (TTR) through MALDI-TOF.

Figure 9

Box plot of transthyretin concentration measured by ELISA. Levels of transthyretin protein (mean±SE) were plotted for control subjects (CO) and patients with primary open-angle glaucoma (POAG). The protein was detected using a specific ELISA. The POAG group showed significantly higher levels of TTR then the control group (p=0.03).