Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits

Abstract

Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.

Increased Glial Fibrillary Acid Protein and Vimentin in Vitreous Fluid as a Biomarker for Proliferative Vitreoretinopathy

“…16 Neither GFAP nor vimentin was reported as a major protein from the vitreous body of nondiseased organ donor eyes in the study by Wu et al 17 Vimentin, but not GFAP, was expressed in healthy rabbit vitreous. 18 For ethical reasons, it is not possible to obtain fresh human vitreous samples from healthy eyes. Therefore we compared elevated vitreous GFAP and vimentin from retinal detachment with vitreous GFAP and vimentin from eyes with macular hole or epiretinal membrane groups.…”

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin

“…Qualitative analysis of the dystrophin-enriched fraction from rabbit skeletal muscle was performed using Proteome Discoverer 1.4 against Sequest HT (SEQUEST HT algorithm, licence Thermo Scientific, registered trademark University of Washington, USA) using the UniProtKB database with 24,202 proteins. Since the rabbit genome is incomplete, mass spectrometry raw files were searched against both the Oryctolagus cuniculus database and the Mammalia database (Liu et al 2016). For proper protein identification, the following search parameters were used: (1) peptide mass tolerance set to 10 ppm, (2) MS/MS mass tolerance set to 0.02 Da, (3) up to two missed cleavages, (4) carbamidomethylation set as a fixed modification and (5) methionine oxidation set as a variable modification.…”

Mass spectrometric identification of dystrophin, the protein product of the Duchenne muscular dystrophy gene, in distinct muscle surface membranes

“…The raw data files from the mass spectrometric analysis were searched against the Oryctolagus cuniculus database. However, the rabbit genome is incomplete and we therefore had to supplement the data analysis by also screening the Mammalia database, as previously described by Liu et al ( 54 ).…”

“…The following search parameters were used for protein identification: i) peptide mass tolerance set to 10 ppm; ii) MS/MS mass tolerance set to 0.5 Da; iii) up to two missed cleavages; iv) carbamidomethylation set as a fixed modification; and v) methionine oxidation set as a variable modification ( 14 ). Since the rabbit genome is incomplete, mass spectrometry raw files were searched against both the Oryctolagus cuniculus database and the Mammalia database ( 54 ). Peptides were filtered using a minimum XCorr score of 1.5 for 1, 2.0 for 2, 2.25 for 3 and 2.5 for 4 charge states, with peptide probability set to high confidence.…”