Spatiotemporal profiling of modification-specific proteome secretion uncovers an itaconation-activated tyrosine kinase

Abstract Macrophages secrete diverse signaling proteins critical for intercellular communication and immune responses, processes tightly regulated by post-translational modifications (PTMs). Itaconate, an immunoregulatory metabolite produced in macrophages, induces widespread intracellular protein modification (itaconation), affecting pathways like the KEAP1-NRF2 axis and glycolysis. However, the impact of itaconation on the extracellular proteome and signaling remains poorly characterized. Herein, we introduce PTM-based secretome profiling (PBSP), a novel approach to identify secreted proteins bearing specific PTMs. The method employs a bioorthogonal probe to label modified proteins in live cells, followed by enrichment of labeled proteins from the culture medium upon secretion. We established a streamlined chemoproteomic workflow integrating spintip-based affinity purification (FISAP) with data-independent acquisition (DIA) mass spectrometry for enhanced sensitivity and coverage. This identified 818 macrophage-secreted itaconated proteins, among which 447 are exosome-dependent. Further biochemical analysis revealed that itaconation of Cys239 on FYN (a tyrosine kinase) enhances its kinase activity in macrophages. We finally demonstrate PBSP’s versatility by profiling secreted proteins with other PTMs, including fumarate-induced succination. PBSP provides a powerful platform to explore PTM roles in protein secretion, offering insights into PTMs’ regulatory functions in cell-cell communication.