Optimized Azidohomoalanine labeling protocol enables sensitive detection of <i>de Novo</i> protein synthesis in <i>C. elegans</i> under heat shock

Living organisms continually adjust protein expression in response to environmental stimuli. To systematically characterize this regulation, we analyzed the C. elegans nascent proteome using L-azidohomoalanine (AHA), a noncanonical methionine analog. Applying AHA to profile de novo protein synthesis during heat shock revealed stimulus-dependent changes in nascent protein production. Although AHA has been used previously to label newly synthesized proteins (NSPs) in C. elegans and enable bioorthogonal tagging via click chemistry, the labeling process itself has not been thoroughly optimized, limiting comprehensive proteomic analysis. Here, we present a modified exploratory study protocol—distinct from that reported in the literature—that improves AHA incorporation by optimizing the metabolic labeling of the E. coli food source. Specifically, inducing E. coli into AHA-containing medium at an OD of 0.2 (logarithmic phase), rather than the conventional OD 1.0 (stationary phase), significantly enhanced AHA labeling efficiency in C. elegans . This optimization increased AHA incorporation into the nascent proteome and yielded stronger post-click-chemistry affinity tag signals, enabling a more complete characterization of protein-regulation dynamics during heat shock. These exploratory findings define improved environmental conditions for efficient bioorthogonal labeling and provide an optimized workflow for future studies. Subsequent exploratory experiments demonstrated reliable in vivo identification of NSPs in C. elegans , supporting sensitive and direct analysis of protein synthesis under varying environmental stimuli.