Research note: Phosphoproteomic analysis of macrophage immunity stimulated by egg white glycopeptide

Egg white glycopeptides (EWG) are dietary peptides with immune regulatory activities; however, the underlying immune regulation mechanisms of EWG-mediated have not been fully elucidated. Phosphorylation represents a crucial protein modification in immune cell signaling. In this study, phosphoproteomic technology was employed to investigate alterations in macrophage phosphoprotein profiles in response to EWG stimulation (EWG group) using lipopolysaccharide stimulation as a positive control (LPS group), and bioinformatics approaches were used to analyze the associated signaling pathways. The untreated macrophages served as the control group (CK group). Compared with the CK group, 625 differentially phosphorylated proteins (DPPs) were identified in the EWG group, involving 774 differential phosphorylation sites. Among them, the number of up-regulated DPPs was less than that of down-regulated DPPs, whereas the LPS/CK group displayed the opposite trend. This finding indicated that the mechanisms underlying EWG- and LPS-activated macrophage immunity were related to protein dephosphorylation, but the regulatory patterns of protein phosphorylation differed between EWG and LPS. In the EWG/CK group, 60.75% of DPPs were located in the nucleus and annotated to immune signaling pathways such as leukocyte transendothelial migration and Fc gamma R-mediated phagocytosis. Similar to LPS, the TLR4/MyD88/NF-κB pathway was identified to be crucial in the immune regulation of EWG-activated macrophages. This study elucidated the changes in the phosphorylated protein profiles underlying the EWG-mediated activation of macrophage immunity, providing a theoretical basis for developing EWG as an immunomodulatory food bioactive compound.