In-depth immunometabolic profiling by measuring cellular protein translation inhibition via bioorthogonal noncanonical amino acid tagging (CENCAT)

Motivation Extracellular Flux (XF) analysis has been a key technique in immunometabolism research, measuring cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine immune cell metabolic profiles. However, XF analysis has several limitations, including the need for purified adherent cells, relatively high cell numbers, and specialized equipment. Recently, a novel flow cytometry-based technique called SCENITH (Single Cell Energetic metabolism by profiling Translation inhibition) was introduced, which measures the inhibition of cellular protein synthesis as a proxy for metabolic activity in single cells. A limitation of this technique is its reliance on fluorescent staining of intracellular puromycin, a toxic antibiotic. To address this, we propose an alternative approach using biorthogonal noncanonical amino acid tagging (BONCAT) to measure protein synthesis.