THRONCAT: Efficient metabolic labelling of newly synthesized proteins using a bioorthogonal threonine analogue

Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insight into cellular physiology. Existing metabolic protein labelling approaches based on bioorthogonal methionine- or puromycin analogueues allow for the selective visualization and enrichment of the newly synthesized proteins, however, their applications are limited as they require methionine-free conditions or are toxic to cells. Here, we introduce a novel threonine-derived non-canonical amino acid tagging method, THRONCAT, based on bioorthogonal threonine analogueue β-ethynyl serine (βES) that enables efficient and non-toxic labelling of the nascent proteome in complete growth media within minutes. We used THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells, and drosophila melanogaster and rapidly profiled proteomic changes of Ramos B-cells in response to receptor activation in a time-stamp approach, demonstrating the potential and ease-of-use of the method.