Glaucoma is a degenerative eye disease causing damage to the optic nerve. One of the challenges in studying retinal degeneration is access to human eye samples, so previous researchers have often documented proteomic changes occurring with glaucoma in animal models. However, Funke et al. (2016) turned to human subjects for their work.1 After careful analysis, the team found numerous proteomic changes in the retina resulting from glaucoma.
The researchers used eye samples donated by the Cornea Bank of Rhineland-Palatinate, within the Department of Ophthalmology at the University Medical Center of Johannes Gutenberg University Mainz, in Germany. They purified lyophilized peptides from five glaucoma samples and five matched healthy controls prior to liquid chromatography and mass spectrometry (LC-MS).
The LC system consisted of a Rheos Allegro pump downscaled to a capillary system that included a 30 × 0.5 mm BioBasic C18 column coupled to a 150 × 0.5 mm BioBasic C18 column (both Thermo Scientific) and an inline precolumn filter.
The team connected the LC system to the electrospray ionization source of an LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap mass spectrometer with a low flow metal needle (both Thermo Scientific) to introduce ions. They also used Xcalibur software, revision 2.0.7 SP1, (Thermo Scientific) to process data.
They then compared spectra with the MaxQuant and Swiss-Prot databases. As a result, the researchers identified more than 600 proteins at high confidence (FDR < 1%) in human retina samples.
Funke and colleagues used accurate inclusion mass screening as well as targeted mass spectrometric analysis to further validate a group of selected protein candidates. Immunostaining of the retinal ganglion cell layer provided another level of confidence using laser capture microdissection in porcine and human eye cryosections.
Researchers observed distinct proteomic changes in 10% of proteins encircling mitochondrial and nucleus species. Numerous proteins showed a significant glaucoma-related level change (p < 0.05) or distinct tendency of alteration (p < 0.1).
Among the candidate proteins, the researchers documented proteins involved in cellular development, stress and cell death. They found an increase of stress-related proteins and a decrease of new glaucoma-related candidates: ADP/ATP translocase 3 (ANT3), PC4 and SFRS1-interacting protein 1 (DFS70), and methyl-CpG binding protein 2 (MeCp2)
Additionally, the researchers hope this work will help direct future research projects.
1. Funke, S., et al. (2016) “Glaucoma related proteomic alterations in human retina samples,” Scientific Reports, 6(29759), doi: 10.1038/srep29759.