TLR4 is expressed on the endosomal membranes of innate immune cells, acting as one of the sensors of danger signals. Upon TLR4 activation, signaling cascades are initiated through MyD88-dependent and TRIF-dependent mechanisms. These pathways induce the expression of proinflammatory cytokines and type 1 interferons. However, molecular mechanisms controlling the formation, interaction, and disassembly of protein complexes remain unclear. To figure it out, we have optimized limited proteolysis-coupled mass spectrometry (LiP-MS) using quantitative proteomics analysis, following a time-course LPS stimulation in immortalized mouse macrophages and subcellular fractionation into cytoplasm and nuclear. Conformational changes in protein dynamics were observed mainly in the cytoplasmic fraction after 1 hour of LPS stimulation. Within the TLR signaling pathway, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKb) displayed significant conformational changes. In the protease degradation assay, IKKb and Elongator complex protein 1 (Elp1) showed a degradation pattern change between the control and LPS stimulation groups. Elp1 is predicted to act as a scaffold protein that assembles active IKK-MAP3K14 complexes based on protein sequence similarity. Our research indicates that IKKb may be involved in so far unidentified interactions with uncharacterized binding partners, indicating novel mechanisms of innate immunity which we are currently investigating. This research was supported by the Division of Intramural Research of NIAID, NIH.