Abstract
Purpose :
Diverse groups of proteins play integral roles in both the physiology and pathophysiology of the retina. However, thorough proteomic analyses of retinae of experimental species are currently unavailable. Here we comprehensively characterize the retinal proteome of a prototypical lab mouse using a discovery-based mass spectroscopy (MS) approach.
Methods :
Retinae from 3 male and 3 female 30-week-old outbred Swiss-Webster ND4 mice were harvested and immeditely processed for MS analysis on a Thermo Fisher (TF) Fusion Orbitrap Mass Spec. Mice were maintained for 3 months in our institutional vivarium. MS1 scans were performed utilizing a resolution of 240,000. MS2 scans used High Energy Collision Dissociation of 30% with resolutions of 30,000. Static modification included carbamidomethyl on cysteines (=57.021), and dynamic modification of oxidation of methionine (=15.9949). Parent ion tolerance was 10ppm, fragment mass tolerance was 0.6Da, and the maximum number of missed cleavages was set to 2. Three technical repeats were ran for each biological sample. We used Proteome Discoverer 2.2 SEQUEST HT scoring for analysis. The reference protein FASTA database was M. musculus. For characterization, only high scoring peptides were considered utilizing a false discovery rate (FDR) of 1%. Downstream analysis utilized Panther and TF classification systems.
Results :
4,634 different proteins were identified and segregated into 26 major protein classes, 9 functional molecular classes, and 12 categories of biological processes. The 5 largest protein classes include: Nucleic Acid Binding (17%), Hydrolases (13%), Enzyme Modulators (10%), Transferases (10%), and Cytoskeletal (6%). Binding and catalytic proteins contributed to 81% of the molecular function class at 39% and 42% respectively. Cellular processing and metabolic processes contributed the most to biological activity at 31% and 25% respectively. The overwhelming diversity of retinal protein functionality was reflected through further analyses revealing 2,086 unique pathway hits across 241 different pathways.
Conclusions :
Using this high-throughput technique, we have unraveled the diverse proteome of the retina, thus providing the most comprehensive proteomic profile known to date. These findings provide a “springboard” for future studies, facilitating the elucidation of the functional relevance of these proteins to the molecular and cellular pathologies that underlie retinal disease.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.