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Phosphoproteome Analysis in Immune Cell Signaling
Deepali Rathore1, Aleksandra Nita-Lazar1
1Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Current Protocols in Immunology
|September 16, 2020
Summary
This study presents a mass spectrometry method to analyze global phosphorylation changes in immune cells upon toll-like receptor (TLR) stimulation. It quantifies phosphoproteome alterations, aiding understanding of immune signaling pathways in macrophages.
Area of Science:
- Immunology and Molecular Biology
- Proteomics and Mass Spectrometry
Background:
- Immune cell signaling relies heavily on protein phosphorylation.
- Toll-like receptor (TLR) activation by pathogen ligands initiates immune responses influenced by phosphoprotein levels.
Purpose of the Study:
- To describe a mass spectrometry-based approach for global phosphoproteome analysis.
- To identify and quantify phosphoprotein changes in immune cells stimulated via TLRs.
- To provide a methodology applicable to various immune cells and stimuli.
Main Methods:
- Stable Isotope Labeling of Amino acids in Cell culture (SILAC) for metabolic labeling.
- Ligand stimulation of TLRs (TLR2, TLR4, TLR7) in mouse macrophages.
- In-solution trypsin digestion, phosphopeptide enrichment, and LC-MS/MS analysis.
Main Results:
- Detailed protocols for phosphoproteome analysis are presented.
- The method allows for quantitative assessment of phosphorylation changes.
- The approach is demonstrated using TLR-stimulated mouse macrophages.
Conclusions:
- Global phosphoproteome analysis is crucial for understanding complex immune signaling in macrophages.
- The described mass spectrometry technique provides a robust method for studying immune cell signaling pathways.
- This methodology is adaptable for diverse immune cell types and soluble stimuli.
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