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In vivo dual-tagging proteomic approach in studying signaling pathways in immune response
Tianyi Wang1, Sheng Gu, Tapani Ronni
1MS M888, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Insights
Researchers developed a novel in vivo dual-tagging method to identify low-abundance signaling molecules in immune cells. This approach successfully identified new MyD88-interacting proteins, including FLAP-1, an activator of NF-kappaB, advancing innate immune response understanding.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Identifying low-abundance, dynamic signaling molecules in immune cells presents significant challenges.
- Existing methods often fail to capture weakly interacting proteins crucial for cellular signaling.
Purpose of the Study:
- To develop and validate a novel in vivo dual-tagging quantitative approach for identifying signaling complex components in immune cells.
- To uncover novel proteins interacting with MyD88 in macrophages upon lipopolysaccharide stimulation.
Main Methods:
- Integration of epitope-tagging for real-time single affinity purification of natural complexes.
- Application of amino acid-coded mass tagging (AACT) for mass spectrometry-based quantitative measurement.
- Development of a dual-tagging method with higher sensitivity and accuracy than traditional tandem affinity purification.
Main Results:
- Successfully identified multiple proteins interacting with MyD88, a key adaptor in innate immunity, in stimulated macrophages.
- Discovered FLAP-1 as a novel activator of NF-kappaB, a critical transcription factor in immune response.
- Demonstrated the efficacy of the dual-tagging approach in preserving weakly interacting molecules.
Conclusions:
- The developed in vivo dual-tagging method enables comprehensive analysis of signaling pathways in immune cells.
- This approach provides global insights into the functional links between MyD88 and its interacting partners in TLR-mediated signal transduction.
- The method is broadly applicable for in vivo analysis of diverse signaling pathways beyond innate immunity.
Abstract:
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.
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