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Proteomic Profiling of Macrophages by 2D Electrophoresis
Published on: November 4, 2014
Profiling of membrane proteins from human macrophages: comparison of two approaches
Marie-Christine Slomianny1, Annabelle Dupont, Fatiha Bouanou
1CNRS UMR 8576, and Université des Sciences et Techniques de Lille, Villeneuve d'Ascq, France.
Abstract:
Macrophages are involved in various important biological processes and their functions are tightly regulated. Hydrophobic proteins are difficult to analyse by 2-DE because of their intrinsic tendency to self-aggregate during the first dimension (IEF). We have compared two protocols for extracting, separating and identifying membrane proteins from human macrophages by MALDI-TOF MS. The first protocol used protein extraction by solvent, followed by 2-DE and allowed us to identify 10% membrane proteins among the proteins identified a being like the peroxisome-activated receptor delta. The second method is based on solubilizing the membranes with Triton X-100, separating the proteins by anion-exchange chromatography followed by SDS-PAGE. This method allowed us to identify 49 membrane proteins, including four integral membrane proteins, ten type I, two type II and one type III membrane proteins. Several receptors were identified, including integrin alpha-3 and ephrin type A receptor 7. Interestingly, several proteins involved in macrophage functions were identified, such as integrin alpha-X and macrophage mannose receptor. These findings show that techniques are available to identify membrane proteins, but that they require large quantities of cells which means that they are not suitable for the limiting amounts of precious samples available from clinical studies.
Insights
Identifying macrophage membrane proteins is challenging due to aggregation. A Triton X-100 method successfully identified 49 membrane proteins, outperforming solvent extraction for macrophage analysis.
Area of Science:
- Cell Biology
- Proteomics
- Immunology
Background:
- Macrophages play crucial roles in biological processes and immune regulation.
- Analyzing hydrophobic membrane proteins is difficult due to self-aggregation during techniques like 2-DE (two-dimensional electrophoresis).
Purpose of the Study:
- To compare two protocols for extracting, separating, and identifying membrane proteins from human macrophages.
- To evaluate methods for overcoming hydrophobic protein aggregation in proteomic analysis.
Main Methods:
- Protocol 1: Solvent extraction followed by 2-DE and MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry).
- Protocol 2: Membrane solubilization with Triton X-100, anion-exchange chromatography, SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), and MALDI-TOF MS.
Main Results:
- Protocol 1 identified only 10% membrane proteins, including peroxisome-activated receptor delta.
- Protocol 2 identified 49 membrane proteins, including integral, type I, II, and III membrane proteins.
- Key receptors (integrin alpha-3, ephrin type A receptor 7) and macrophage function proteins (integrin alpha-X, macrophage mannose receptor) were identified.
Conclusions:
- The Triton X-100 based method is superior for identifying a broader range of membrane proteins in macrophages.
- Current techniques require large cell quantities, limiting their application to precious clinical samples.
