In vivo dual-tagging proteomic approach in studying signaling pathways in immune response.
Up to date, few successes have been achieved to identify the signaling molecules directly from immune cells due to their low-abundance and dynamic nature. Here, we designed an in vivo dual-tagging quantitative approach that integrated epitope-tagging which allows single affinity purification of the natural complexes formed at real-time, and amino acid-coded mass tagging (AACT) that assists mass spectrometry-based quantitative measurement, to identify the specific components of a signaling complex formed in macrophage cells upon lipopolysaccharide (LPS) stimulation. The sensitivity and accuracy of this quantitative method are significantly higher than those of tandem affinity purification, because the multiple step of purifications are avoided to preserve weakly interacting molecules. We identified a number of proteins that interact with MyD88, a critical adaptor protein in innate immune response, in macrophages upon stimulation. Among those newly identified MyD88-interacting partners, FLAP-1 was found to be an activator of NF-kappaB, the key transcription factor in immune response. This integrated approach provides global information on the functional link between MyD88 and other proteins in transducing the TLR-mediated signal and is generally applicable to in vivo analyses of other signaling pathways.
Wang
Tianyi
T
MS M888, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Gu
Sheng
S
Ronni
Tapani
T
Du
Yu-Chun
YC
Chen
Xian
X
D058927:5-Lipoxygenase-Activating Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D002352:Carrier Proteins; D002460:Cell Line; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology*; D013058:Mass Spectrometry; D008565:Membrane Proteins; D051379:Mice; D015336:Molecular Probe Techniques; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D011971:Receptors, Immunologic / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*
D058927:5-Lipoxygenase-Activating Proteins; D048868:Adaptor Proteins, Signal Transducing; C548593:Alox5ap protein, mouse; D000943:Antigens, Differentiation; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D011971:Receptors, Immunologic