Time-Resolved Proteomic Analysis in Zebrafish Using Bioorthogonal Noncanonical Amino Acid Tagging.

Tracking time-dependent proteomic changes remains challenging. Bioorthogonal noncanonical amino acid tagging (BONCAT) offers a targeted approach for identifying proteins synthesized within defined time windows. Here, we describe BONCAT proteomic analysis in larval zebrafish, a model organism widely used for its genetic tractability and utility in developmental biology and neuroscience. We enriched and identified azidohomoalanine-labeled proteins via mass spectrometry after labeling durations as short as 12 h, achieving significant signal above background compared to unlabeled controls. As a proof of concept, we investigated proteomic changes in response to heat shock. BONCAT analysis revealed upregulation of heat shock-induced proteins with greater sensitivity than global proteomics. Gene set enrichment analysis confirmed that heat shock response proteins were significantly enriched in BONCAT samples but not in whole lysates, highlighting BONCAT's ability to detect otherwise-masked transient molecular responses. Beyond the expected changes in synthesis of heat shock proteins, BONCAT identified differentially expressed proteins implicated in stress responses, lipid metabolism, and neural regulation, offering insights into the zebrafish heat shock response. These findings establish BONCAT as a powerful tool for time-resolved proteomic analysis in zebrafish, and for elucidation of the molecular underpinnings of behavior, stress responses, and development in this versatile model organism.