Within this PoC we performed three main research actions:
1) Undertake large-scale synthesis of metabolic label β-ethynyl serine (βES) on gram scale.
Previous synthetic route reported in our original publication (Bob J. Ignacio, Jelmer Dijkstra, Erik Slot, Natalia Mora Garcia, Margot van Weijsten, Erik Storkebaum, Michiel Vermeulen, Kimberly M. Bonger* THRONCAT: Metabolic labelling of newly synthesized proteins using a bioorthogonal threonine analogue. Nat. Commun. 2023, 14, 3367) involved expensive catalysts and due to moderate reaction yields deemed unsuitable for large scale implementation and commercialisation. The work performed within this PoC involved the development of a new synthetic route to obtain large quantities (multi-gram) of βES that we have accomplished.
2) established a proof of concept for secretome profiling with βES in a therapeutically relevant model system.
During the PoC, and in collaboration with other laboratories, we have optimized the enrichment of secretory proteins of a clinically relevant model, including primary cells isolated from healthy blood donors as well as ex vivo tumor cells. The secretome proteins were successfully isolated by pull down protocols and identified by high-resolution mass spectroscopy analysis. A significant effort was undertaken to validate that βES did not affect cell fitness and behavior. We have identified the uptake receptors of βES in a tumor cell line and validated that βES behave similarly as threonine.
3) demonstrate the detection of drug-induced proteome changes in a relevant and well characterized cell line as well as labeling proteins in an in vivo mouse model.
Protein synthesis is highly dependent on energy provided by ATP which can be produced via different mechanisms within the cell. Cell activity may alter the route of ATP production depending on the stimulus and can serve as proper readout. In collaboration, we previously validated that cell activity can be profiled by inhibiting the independent ATP production mechanisms (Vrielink et al. CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging. Cell Reports Methods, 2024, 4(10), 100883). Due to the increased recognition of immune cell metabolism in various diseases we have extended on this within this PoC. We have labeled cells in vivo using βES and profile all tumor- as well as immune cells within the tumor on their activity and ATP dependence measured by flow cytometry, a faster and more high-thoughput method compared to high resolution mass spectrometry. We found that cell activity can be quantified on single cell level using flow cytometry, which is a promising approach to predict cell response to drug treatment and resistance.
