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Proteomic approach to the identification of cell membrane proteins
H Watarai1, Y Inagaki, N Kubota
1Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Takasaki, Gunma, Japan.
Abstract:
The expression of plasma membrane proteins in human monocyte-derived U937 cells was examined by cell disruption and isolation of microsomal fractions. Two alternative procedures for cell disruption, Dounce homogenization and nitrogen cavitation, were compared. Cell homogenization and sequential centrifugation resulted in an approximately fivefold enrichment of plasma membrane proteins in the microsomal fraction. However, identification of 30 such apparently enriched proteins by two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry revealed that only eight were plasma membrane proteins, the remaining 22 being contaminants. In contrast, nitrogen cavitation followed by sequential centrifugation and solubilization of proteins with sodium dodecyl sulfate (SDS) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) detergent yielded subcellular fractions, including microsomes, that showed little overlap in constituent proteins as revealed by 2-D electrophoresis. These results highlight the importance of obtaining pure plasma membranes and complete solubilization of membrane proteins for proteomic analysis.
Insights
Comparing cell disruption methods for U937 cells, nitrogen cavitation yielded purer plasma membrane protein fractions than Dounce homogenization for proteomic analysis.
Area of Science:
- Cell Biology
- Proteomics
- Biochemistry
Background:
- Plasma membrane proteins are crucial for cellular functions.
- Accurate isolation of plasma membranes is essential for proteomic studies.
- U937 cells are a common model for monocyte research.
Purpose of the Study:
- To compare two cell disruption methods for isolating plasma membrane proteins from U937 cells.
- To evaluate the purity of isolated fractions using proteomic techniques.
- To optimize methods for plasma membrane protein analysis.
Main Methods:
- Human monocyte-derived U937 cells were used.
- Two cell disruption techniques were compared: Dounce homogenization and nitrogen cavitation.
- Subcellular fractions were isolated via sequential centrifugation.
- Protein identification was performed using two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry.
Main Results:
- Dounce homogenization followed by centrifugation enriched plasma membrane proteins fivefold but contained significant contaminants (22 out of 30 identified proteins).
- Nitrogen cavitation followed by centrifugation and detergent solubilization (SDS and CHAPS) yielded purer subcellular fractions with less protein overlap.
- 2-D electrophoresis revealed distinct protein profiles between fractions obtained by the two methods.
Conclusions:
- Nitrogen cavitation is a more effective method for isolating purer plasma membrane fractions from U937 cells compared to Dounce homogenization.
- Complete solubilization of membrane proteins is critical for comprehensive proteomic analysis.
- These findings emphasize the importance of optimized sample preparation for accurate plasma membrane proteomics.