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Lei Zhang, Cheri Stowell, Geeng-Fu Jang, Jack S Crabb, Belinda Willard, Claude Burgoyne, John W Crabb; Quantitative Proteomic Analysis Of Non-Human Primate (NHP) Optic Nerve (ON) In Early Experimental Glaucoma (EEG). Invest. Ophthalmol. Vis. Sci. 2014;55(13):4519.
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© ARVO (1962-2015); The Authors (2016-present)
To study the molecular mechanisms underlying ON alterations in NHP EEG and discover candidate blood-borne biomarkers, we quantified proteomic changes in the ON of Rhesus Macaques with laser-induced, unilateral EEG.
A 3-6 mm piece of orbital ON (starting about 2 mm behind the globe globe) adjacent to the globe was isolated from the EEG and control eyes of 4 adult NHP (3 high IOP (IOP Max >28 mm Hg, and 1 low IOP (IOP Max < 20 mm Hg). Protein was extracted in SDS and digested with trypsin. Peptides were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Proteins were identified using the Swiss-Protein human database; iTRAQ tags were quantified by the weighted averaged method. Proteins were significantly altered if average ratios (EEG/control) determined with ≥ 2 unique peptides in the 3 high IOP animals (or in the low IOP animal) were at least 1 standard deviation from the mean with p values ≤ 0.05. Proteomic analysis of EEG and POAG plasma was performed in separate experiments following immunodepletion. Bioinformatics analyses utilized Ingenuity Pathway Analysis.
Within the high IOP ON, 54 significantly altered proteins were identified within 944 proteins quantified. Altered proteins were often different in the low IOP ON. Significantly elevated proteins in high IOP NHPs included cytosol aminopeptidase, Ras GTPase-activating-like protein IQGAP1, peripherin and actin-related protein2/3 complex 4. Significantly decreased proteins in high IOP NHPs included protein S100-B, dynamin-1, tubulin beta-3, and endophilin-A1. Many of the altered proteins were detected in NHP EEG plasma, human POAG plasma, and the human plasma atlas, supporting the possibility of monitoring these proteins for biomarker validation.
Top canonical pathways associated with altered ON proteins in high IOP NHP EEG included actin cytoskeletal signaling, epithelial adherens junction signaling/remodeling and gap junction signaling. Top networks included cellular assembly, organization and movement. While further analyses are required, proteomic differences in ON appear to exist between low and high IOP NHPs. Altered proteins provide insights into the molecular mechanisms underlying early ON alterations in NHP EEG and candidate blood-borne biomarkers.
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