Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish.

ABSTRACT

Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce non-canonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells' own translational machinery. Using the selective 'click reaction', this allows for the identification and visualization of newly synthesized proteins in vitro. Here we demonstrate that non-canonical amino acid labeling can be achieved in vivo in an intact organism capable of simple learning behavior, the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, stimulation with a GABA antagonist that elicits seizures in the larval zebrafish causes an increase in protein synthesis throughout the proteome, which can also be visualized in intact larvae.