Combining activity-based protein profiling and flow cytometry to elucidate the role of lipid-metabolizing enzymes in cell heterogeneity

Abstract

Abstract Cell metabolism is mediated by intracellular enzymes that supply essential and specific molecular cues needed to initiate the differentiation of homogenous cell populations into heterogenous cell populations. Intracellular enzymes involved in the metabolism of lipids are of particular interest as lipids play key roles in cell signaling and differing lipid-metabolizing enzyme activity may produce varying signaling profiles, and thus, a varying cell phenotype. To understand how these enzymes regulate function at the single-cell level, we aim to develop methodology that combines activity-based protein profiling (ABPP) and flow cytometry to allow for the separation of heterogenous cells into new populations based on enzyme activity. Our initial studies have focused on targeting serine hydrolases, as this family of enzymes includes numerous intracellular lipases involved with the metabolism of lipids. We have synthesized a modified activity-based fluorescent probe containing a triazole urea warhead known to label active serine hydrolases in live cells. Based on the fluorescent profile generated by measuring single cell fluorescence using flow cytometry, the probe targeted a select population of cells when compared to a control probe and unlabeled cells. This probe-labeled population was concentration dependent as higher concentration of probe labeled more enzyme, suggesting that a different population of cells could be collected between two different concentrations. After these cells are sorted based on the differing fluorescent signals, these new subsets will be analyzed via targeted lipidomics and proteomics methods to quantity any differences in the probed intracellular enzyme expression. This work was supported by the National Science Foundation CAREER Award no. 1942467.