CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging

Cellular energy metabolism significantly contributes to immune cell function. To further advance immunometabolic research, novel methods to study the metabolism of immune cells in complex samples are required. Here, we introduce CENCAT (cellular energetics through noncanonical amino acid tagging). This technique utilizes click labeling of alkyne-bearing noncanonical amino acids to measure protein synthesis inhibition as a proxy for metabolic activity. CENCAT successfully reproduced known metabolic signatures of lipopolysaccharide (LPS)/interferon (IFN)γ and interleukin (IL)-4 activation in human primary macrophages. Application of CENCAT in peripheral blood mononuclear cells revealed diverse metabolic rewiring upon stimulation with different activators. Finally, CENCAT was used to analyze the cellular metabolism of murine tissue-resident immune cells from various organs. Tissue-specific clustering was observed based on metabolic profiles, likely driven by microenvironmental priming. In conclusion, CENCAT offers valuable insights into immune cell metabolic responses, presenting a powerful platform for studying cellular metabolism in complex samples and tissues in both humans and mice. [Display omitted] •Immunometabolic profiling of complex samples and tissue-resident immune cells•Utilizes ncAAs to measure protein translation inhibition without toxic side effects•CENCAT reproduces known metabolic signatures of macrophage activation•βES is the preferred ncAA for CENCAT due to its superior incorporation rate Extracellular flux (XF) analysis has been a key technique in immunometabolism research, measuring the 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 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 that could cause unwanted effects during the assay. To address this, we propose an alternative approach using biorthogonal noncanonical amino acid tagging (BONCAT) to measure protein synthesis. Vrieling et al. present