Phosphoproteomic profiling of lipopolysaccharide stimulated toll-like receptor pathways in macrophages

Toll-like receptors (TLRs) are present on the surface of immune cells such as dendritic cells, macrophages, and natural killer cells. TLRs and other pattern recognition receptors (PRRs) are essential for the recognition of microbiological components. They trigger both innate and adaptive immune responses to defend against pathogenic microorganisms. Among the TLRs, TLR4 is one of the best-studied; it recognizes lipopolysaccharide (LPS) and initiates both TRIF- and MyD88-dependent signaling cascades. Regulators of TLR4 signaling, including numerous protein kinases, have a key role in innate and adaptive immune responses. Although TLR4 signaling pathways have been investigated using phosphoproteomics by mass spectrometry using data-dependent acquisition (DDA), the phosphoprotein landscape of the TLR4 signaling remains poorly incomplete, partly due to the technical limitations of DDA. To address this, we utilized data-independent acquisition (DIA) mass spectrometry to deeply explore phosphorylation dynamics within the LPS-stimulated TLR4 signaling pathway in macrophages.