Introduction: Mapping protein dynamics during development is key to elucidating the mechanisms underlying protein regulation in healthy and diseased developing tissues. However, the development of protein labeling techniques that enable such analysis remains lacking. To address this gap, we developed a technique that enables labeling nascent murine embryo proteome via injecting the non-canonical amino acid azidohomoalanine (Aha) into pregnant mice. Aha is an azide-modified methionine analog that is incorporated into nascent proteins using endogenous translational machinery. The azide group of Aha allows selective enrichment of nascent proteins via click chemistry using alkyne-bearing tags. Using this technique, we were able to study protein regulation during embryonic development. Additionally, this technique enabled probing the rate of synthesis and turnover of intra- and extra-cellular proteins in embryos. Methods: Mice were injected with Aha at different embryonic time points. Embryos were homogenized and Aha-labeled proteins were clicked to biotin-alkyne. Biotinylated proteins were enriched with NeutrAvidin resin, and analyzed with LC–MS/MS. For turnover analysis, embryos were harvested at different time points following Aha injection, intra- and extra-cellular proteins were fractionated based on solubility using buffers of increasing stringency, and analyzed by western blotting. Results: Analysis of nascent proteins enriched at different developmental time points indicated the enrichment of proteins that play roles in biological processes occurring around each developmental time point. Additionally, turnover studies showed that protein labeling occurs within 3h after Aha injection and that maximum labeling is around 6h. Interestingly, the analysis revealed that extra-cellular proteins have significantly lower turnover rates than intra-cellular proteins. Conclusions: The developed method allows selective labeling and enrichment of nascent intra- and extra-cellular proteins in developing mice. Additionally, this method enables quantifying the rates of protein synthesis and turnover during development. Ultimately, mapping protein dynamics during development will significantly impact our understanding of protein regulation in health and disease.

Probing intra- and extra-cellular proteins dynamics in vivo using non-canonical amino acid tagging

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.