Integrated Proteomic Profiling of Whole-Cell and Multi-Post-Translational Modifications Unveils Regulatory Networks...

The RAW264.7/THP1 cells have been extensively employed as macrophage models in inflammation research. However, comprehensive comparisons of their expression profiles remain largely unexplored. In this study, we conducted a systematic bioinformatics analysis to characterize the whole-cell proteome, phosphoproteome, acetylome, and ubiquitinome profiles of lipopolysaccharide-stimulated RAW264.7/THP1 cells. Through comparative temporal analysis of differentially regulated proteins (classified as rapid, persistent, or slow dysregulation patterns), we identified IGF2, COPZ1, and GTF2B, exhibiting persistent downregulation in both cell models. Additionally, we observed that nearly all dysregulated proteins within each modification type exhibited significant enrichment in the other two modification types, forming a highly interconnected protein-protein interaction network. Notably, VIMENTIN demonstrated significant downregulation across three modification types (phosphorylation at Ser56/214, acetylation at Lys235, and ubiquitination at Lys139), with coordinated downregulation observed in both cell lines. It was also shown that VIMENTIN mutation at Ser56, Lys235, and Lys139 dramatically reduces the expression of CDK1, which may help the inflammation response by averting mitotic catastrophe and changing the macrophage cell cycle. These findings provide valuable molecular insights into species-specific macrophage responses and establish an important reference data set for discovering novel inflammatory proteins and investigating macrophage-associated diseases using these experimental models. Notably, our work delineates vimentin-centered regulatory networks that may offer novel diagnostic biomarkers and therapeutic targets for inflammatory diseases.