Proteome-Wide Analysis of Palmitoylated Proteins in Macrophages Reveals Novel Insights Into Early Immune Signaling

Protein S-palmitoylation, a reversible lipid modification, plays critical roles in regulating protein function and localization. However, its comprehensive role in the rapid reprogramming of macrophages during early immune responses remains incompletely understood. This study investigates the dynamics of the palmitoylome in immortalized bone marrow-derived macrophages (iBMDMs) during the initial phase of lipopolysaccharide (LPS) stimulation. Employing acyl-biotin exchange (ABE) proteomics coupled with a multi-protease digestion strategy (trypsin, AspN, chymotrypsin, or GluC), we significantly enhanced palmitoylation proteome coverage, identifying 2502 putative S-palmitoylated proteins (Log₂ fold change > 2, FDR < 0.05). Notably, this approach uncovered 527 proteins not previously associated with the mouse palmitoylome, including 185 candidates exclusively identified using non-tryptic proteases. In the context of immune cells, this study revealed 1378 proteins not previously reported, with 556 candidates identified exclusively via AspN, chymotrypsin, and/or GluC. Several of these novel candidates are established immune system components and phosphoproteins. Upon stimulation with 100 ng/mL LPS for 30 min, quantitative comparison revealed 648 differentially enriched proteins (308 predominantly detected in untreated, 340 predominantly detected in LPS-treated), indicating dynamic regulation via this posttranslational modification during early innate immune activation. Functional enrichment analysis linked these dynamically regulated proteins to critical pathways: LPS treatment enriched for immune signaling cascades and infection pathways, while untreated cells showed enrichment for metabolic and transport processes. This study provides a comprehensive resource of the macrophage palmitoylome and its dynamic remodeling, offering novel targets for investigating the regulation of macrophage function.