Abstract
Purpose :
Understanding the spatial and temporal expression pattern of different proteins in each of the ~100 retinal cell types provides essential information about the dynamic regulation of diverse processes in the retina under normal and diseased conditions. However, analysis of the retinal proteome is limited by the inability to apply a single type of chemistry to label proteins for subsequent purification and analysis. Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) may provide a solution to that problem. The goal of this project is to test the efficiency of applying BONCAT to the mouse retina.
Methods :
Azidohomoalanine (AHA) is a non-canonical amino acid with an azide functional group that is used as a methionine substitute. AHA, or PBS as the negative control, was delivered to C57Bl6/J mice (male and female; age 1-6 months) by intraperitoneal injection. Mice were fed either normal chow or a low methionine diet. A range of AHA doses (2-10 mg per 20 g body weight) were delivered for 1-4 days. Subsequently, retinas were harvested and stored at -80C. Proteins from stored retinas were extracted using a lysis buffer containing PBS supplemented with 150 mM NaCl, 50 mM Tris (pH 7.4 ), 1% Triton X-100, 0.1% SDS, and a broad-spectrum protease and phosphatase inhibitor cocktail (Roche). AHA containing proteins were labeled using a copper-dependent click reaction (Click-&-Go Protein Reaction buffer kit, Click Chemistry Tools) with alkyne-biotin. Proteins were methanol-chloroform precipitated prior to analysis by western blotting, with or without concentration on neutravidin beads.
Results :
Western blotting using a streptavidin antibody demonstrated labeling of biotin-clicked retina proteins in AHA injected mice with low non-specific labeling in PBS injected mice. Placing mice on a low methionine diet throughout the experiment increased the amount of labeled protein. An AHA dose of 2 mg per 20 g body weight is sufficient. Higher doses of AHA caused toxicity, as seen by liver discoloration or death of the animal. Overall labeling intensity increased with the number of daily AHA injections.
Conclusions :
Systemically delivered AHA is robustly incorporated into mouse retina proteins. Click chemistry can be used to covalently attach biotin tags for subsequent proteomic analysis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.