Dynamic proteome and phosphoproteome profiling reveals regulatory mechanisms in LPS-stimulated macrophage inflammatory responses

Macrophage-mediated acute inflammation is crucial for pathogen clearance and tissue repair, yet the underlying molecular mechanisms remain inadequately understood. The present study focused on the dynamic profiles of the proteome and phosphoproteome of macrophages exposed to lipopolysaccharide within 1 h. Gene Set Enrichment Analysis (GSEA) identified significantly enriched pathways in fatty acid metabolism and translation during the early inflammatory phase. Further trend analysis of the differentially expressed proteins revealed patterns associated with translation regulation such as translation initiation. Importantly, the nascent chain experiment demonstrated no significant changes in overall gene translation levels during this phase. These data indicate that macrophages maintain intracellular protein homeostasis through translational regulation, with post-translational modifications (PTMs) playing a crucial role in the rapid cellular response to pathogen invasion. Phosphorylation is a key PTM that regulates protein functions in almost all cellular processes. Time-resolved phosphoproteome analysis identified 367 differentially expressed phosphopeptides involved in immunerelated pathways that resist infection. Additionally, weighted gene co-expression network analysis (WGCNA) discovered core modules that regulate translation-related processes such as RNA export from nucleus. Moreover, conjoint analysis of the proteome and phosphoproteome identified the hub protein EF1B that exhibited the largest fold change and is also involved in translation. Our data not only provide a more comprehensive understanding of the dynamic molecular networks of acute macrophage inflammation but also provide a systematic proteomic resource for further studies.