Time-Resolved Proteomic Analysis in Zebrafish and Cultured Neurons Using Bioorthogonal Noncanonical Amino Acid Tagging

Temporally and spatially controlled protein synthesis plays a critical role in orchestrating the molecular events underlying behaviors, stress adaptations, and therapeutic responses to drugs. However, traditional proteomic techniques often fail to capture the dynamic changes in protein expression essential for understanding transient biological phenomena. To overcome this limitation, the work presented in this thesis leverages bioorthogonal noncanonical amino acid tagging (BONCAT) coupled with mass spectrometry to perform time-resolved proteomic analyses in zebrafish larvae and cultured neurons. Chapter II details the development and validation of BONCAT proteomics in zebrafish, demonstrating that newly synthesized proteins from zebrafish larvae could be reliably labeled, enriched, and identified even over short labeling periods. Proof-of-concept experiments using heat shock revealed that BONCAT proteomics was able to detect changes in expression of proteins known to be induced by heat shock with greater sensitivity than conventional approaches using global proteomics. These results establish BONCAT as a powerful tool for investigating dynamic changes in protein synthesis in zebrafish. In Chapter III, we applied BONCAT to neuronal cultures to profile the proteomic changes induced by sub-anesthetic, antidepressant-relevant doses of ketamine. These studies uncovered rapid alterations in protein synthesis, identifying significantly differentially regulated proteins and pathways involved in synaptic plasticity, cytoskeletal remodeling, cellular signaling, metabolism, and RNA processing. This work provides novel molecular insights into ketamine’s rapid-acting antidepressant effects and further illustrates the utility of BONCAT for capturing early, transient proteomic responses to drug treatment. Finally, in Chapter IV, we explore changes in protein expression in zebrafish larvae underlying circadian rhythms and in response to low-dose ketamine treatment. We observed interesting protein synthesis patterns in both biological contexts, but our findings lacked the statistical significance and reproducibility across experiments required to draw strong biological conclusions from our data. Although methodological refinements are required, our work underscores BONCAT’s potential to elucidate transient proteomic shifts underlying behavioral phenomena and pharmacological interventions in zebrafish.