Optimizing Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) for low-disruption labeling of Arabidopsis proteins <i>in vivo</i>

Plants require robust responses in protein synthesis to adapt to variable environmental conditions. Measurement of newly synthesized proteins across several organisms has been successfully facilitated with Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT). Here, we use non-canonical amino acids (NCAAs) L-azidohomoalanine (AHA) or L-homopropargylglycine (HPG) incorporation in place of methionine residues into the actively translating Arabidopsis (Arabidopsis thaliana) proteome, allowing for that subset of proteins to be enriched for mass spectrometry quantification. Although this technique has seen occasional use in plants, optimization of the protocol to maximize functionality while minimizing organismal stress remains to be established. Here, we provide evidence for successful implementation through the liquid immersion of seedlings in AHA- or HPG-containing media that functions with significantly lower concentrations than the literature standard. Our approach splits acute exposure and the incorporation phase of labeling to mitigate potential negative impacts of prolonged NCAA exposure without compromising effective enrichment capacity. This method results in an unperturbed growth phenotype for AHA-treated seedlings. Finally, we demonstrate the capacity of this modified approach to enrich newly synthesized proteins from the whole proteome under standard stress conditions. These improvements allow for a broader use of BONCAT technologies in molecular plant research, affording a deeper understanding of the newly synthesized proteome without negatively impacting plant health.