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Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis
Published on: April 17, 2017
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Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis
Kathrin Rösch1, Marcel Kwiatkowski2, Hartmut Schlüter2
1Heinrich Pette Institute, Leibniz Institute for Experimental Virology.
Journal of Visualized Experiments : Jove
|April 28, 2017
Summary
This study details a protocol for analyzing proteins associated with lipid droplets during Hepatitis C Virus (HCV) infection. The method uses quantitative proteomics to identify host factors influencing viral replication.
Area of Science:
- Virology
- Cell Biology
- Proteomics
Background:
- Lipid droplets are crucial for the replication of pathogens like Hepatitis C Virus (HCV), serving as sites for virion assembly.
- Quantitative proteome analysis of lipid droplets can reveal host proteins affected by viral infections.
Purpose of the Study:
- To describe a protocol for characterizing lipid droplet proteome changes during HCV infection.
- To identify host proteins that interact with lipid droplets in response to HCV.
Main Methods:
- Utilized Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) for quantitative proteomic analysis.
- Employed mechanical lysis, differential ultracentrifugation, and washing steps for lipid droplet isolation.
- Analyzed protein purity via western blotting and identified proteins using liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS).
Main Results:
- Successfully identified proteins recruited to lipid droplets following HCV infection.
- These identified proteins may function as pro- or antiviral host factors.
Conclusions:
- The described protocol provides a robust method for studying lipid droplet proteome dynamics.
- This approach is applicable to various cell types and conditions, including pathogen infection, stress, and drug treatment.
