Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish

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 noncanonical 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 noncanonical 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

Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish

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 noncanonical 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 noncanonical 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

State-Selective Metabolic Labeling of Cellular Proteins

Transcriptional activity from a specified promoter can provide a useful marker for the physiological state of a cell. Here we introduce a method for selective tagging of proteins made in cells in which specified promoters are active. Tagged proteins can be modified with affinity reagents for enrichment or with fluorescent dyes for visualization. The method allows state-selective analysis of the proteome, whereby proteins synthesized in predetermined physiological states can be identified. The approach is demonstrated by proteome-wide labeling of bacterial proteins upon activation of the P_BAD promoter and the SoxRS regulon and provides a basis for analysis of more complex systems including spatially heterogeneous microbial cultures and biofilms