Comparative phosphoproteomics studies of macrophage response to bacterial virulence effectors.

Lipopolysaccharide (LPS) from Gram-negative bacteria and cell wall components from Gram-positive bacteria are pathogenic inducers of host cell innate immune systems. In this study, we adapted stable isotope labeling with amino acid in cell culture (SILAC) and Fe(3+)-IMAC phosphopeptide enrichment method to study phosphoproteomic changes in bacterial virulence factors induced macrophage RAW 264.7 cells. In total, we quantified 2657 phosphopeptides and 1990 phosphopeptides in LPS treated and heat-killed Staphylococcus aureus (HKSA) treated macrophage samples respectively. Functional bioinformatics analysis was followed to show differences between LPS and HKSA stimulated macrophage signaling pathways. We identified differences in immune-related signaling networks, including Erk1/2 signaling pathway, Jak/Stat signaling pathway, Jnk signaling pathway, and revealed differences in cytoskeleton reorganization, GTPase regulators and in phosphorylation motifs.