Updated project metadata. Toll-like receptors are among the first sensors that detect and drive immune responses to pathogens. Macrophages that encounter a pathogen are usually not stimulated through one TLR but by a combination of these receptors engaged by distinct ligands produced by the microbe. As a first step to understanding the integrated signaling under such complex conditions, we have investigated the differences in the phosphoprotein signaling cascades triggered by individual TLR4, TLR2 and TLR7 ligands using a single responding cell population. We performed a global quantitative and early post-stimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry.