Synapses are dynamic, with respect to their magnitude of synaptic transmission, their morphologic structure and their protein components. Dynamic changes in the synaptic proteome, mediated by changes in protein synthesis, degradation, distribution and post- translational modifications affect synaptic function and the capacity for synaptic plasticity. Despite the appreciation of the biological importance of synaptic protein dynamics, methods to enrich for and identify the dynamic synaptic proteome are not yet an integral part of neuroscience research. Studies conducted largely in prokaryotes and non-neuronal cells have demonstrated the promise of using the incorporation of non- canonical amino acids into newly synthesized proteins followed by bio-orthogonal click chemistry to tag the non-canonical amino acid labeled protein with probes, such as biotin, that can be used for purification and identification of newly synthesized proteins. We propose experiments to optimize the use of non-canonical amino acid incorporation and bio-orthogonal click chemistry in neuronal cell cultures, and in the visual system of intact rats and Xenopus tadpoles to demonstrate the feasibility of metabolic labeling to identify and quantify dynamic components of the synaptic proteome. These experiments are meant to represent proof of principal for comparing synthesis of synaptic proteins between different conditions and in different experimental systems.
Newly synthesized synaptic proteins regulate synaptic function and plasticity. We propose to improve feasibility of using metabolic labeling with non-canonical amino acids to identify and quantify changes in the synaptic proteome in neuronal cultures and intact animals.
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