Testing the robustness of a BONCAT strategy for detection of rapamycin-induced small changes in protein translation after short-term treatment of HEK293 cells

BONCAT (Bioorthogonal non-canonical amino acid tagging) is widely used for identification of nascent proteins. We aimed to test the robustness of this technique for the investigation of rapamycin-induced changes in protein translation after different incubation times. Initial experiments for enrichment of Azidohomoalanine-tagged proteins showed that in our hands clicking of newly synthesized proteins (NSPs) directly to alkyne agarose outperformed enrichment via alkyne biotin derivatives. We then chose to treat HEK293 cells for six hours with rapamycin, inhibitor of mTOR (Serine/threonine-protein kinase mTOR), as proof of concept for our protocol to specifically enrich for NSPs. After clicking of NSPs to the alkyne agarose, we performed an on-bead tryptic digest and peptides were analyzed by LC-MSMS. MaxQuant analysis resulted in detection of >2600 proteins in total, 451 and 519 proteins uniquely identified in the untreated and treated sample, respectively. As expected, GO Enrichment analysis (PANTHER, ShinyGO) of differentially newly synthesized proteins (|log2(FC)|≥1) could link the results specifically to rapamycin treatment since major known cellular processes are affected, such as ribosome biogenesis and translation as well as splicing. Next, HEK293 cells were treated again with rapamycin but this time for a shorter period of only two hours. With these results we want to demonstrate the suitability of our BONCAT protocol to robustly identify even small changes in protein translation.