Comprehensive characterization of the adult ND4 Swiss Webster mouse retina: Using discovery-based mass spectrometry to decipher the total proteome and phosphoproteome.

ABSTRACT

Purpose: Diverse groups of proteins play integral roles in both the physiology and pathophysiology of the retina. However, thorough proteomic analyses of retinas of experimental species are currently unavailable. The purpose of the present paper is providing the field with a comprehensive proteomic characterization of the retina of a commonly used laboratory mouse using a discovery-based mass spectrometry (MS) approach. Methods: Retinas from eight male and eight female 30-week-old outbred ND4 Swiss Webster mice were harvested and immediately processed for MS analysis on a Thermo Fisher (TF) Fusion Orbitrap MS. The retinal proteome and phosphoproteome were identified and subsequently analyzed using Proteome Discoverer 2.2 and Panther-GeneGo. SEQUEST-HT scoring was used for analysis, and the reference protein FASTA database was from Mus musculus. Specifically, three technical repeats were performed for each biological sample. For characterization, only high-scoring peptides were considered, with a false discovery rate (FDR) of <1%. Downstream bioinformatic analysis used Ingenuity Pathway Analysis (IPA; Qiagen). Results: Using Proteome Discoverer 2.2, 4,767 different proteins were identified and segregated into 26 major protein classes, 9 functional molecular classes, and 12 categories of biological processes. The five largest protein classes included the following nucleic acid binding (17%), hydrolases (13%), enzyme modulators (10%), transferases (9%), and oxidoreductases (6%). "Binding" and "catalytic" proteins contributed to 81% of the molecular function class at 37% and 42%, respectively. "Cellular processing" and "metabolic processes" contributed the most to biologic activity, at 31% and 26%, respectively. Phosphopeptide enrichment yielded the identification of 610 additional unique proteins that were not originally identified. The two datasets combined produced an adult mouse retinal proteome consisting of 5,377 unique proteins. Overall, 41% of the retinal proteome was phosphorylated. The overwhelming diversity of retinal protein functionality was reflected through further analyses revealing 2,086 unique pathway hits across 241 different pathways (TF). A core analysis summary report was performed in IPA (Qiagen) to analyze the top signaling networks, protein-protein interaction (PPI) enrichments, and canonical pathways. Conclusions: Using this high-throughput technique, we have further deciphered and updated the diverse proteome of the mouse retina, including the phosphoproteome, thereby providing the most comprehensive proteomic profile for this tissue known to date. These findings, and the bioinformatic analyses we also provided, establish a platform for future studies, facilitating the elucidation of the relevance of these proteins to the molecular and cellular pathologies that underlie retinal function and disease.