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Proteomic analysis of low-abundant integral plasma membrane proteins based on gels
1Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Science, Hunan Normal University, Changsha, PR China.
Abstract:
To characterize low-copy integral membrane proteins and offer some methods for human liver proteome projects, we fractionated highly purified rat liver plasma membrane (PM). PM was purified through two sucrose density gradient centrifugations, and treated with 0.1 M Na(2)CO(3), chloroform/methanol and Triton X-100. Proteins were separated by electrophoresis and submitted to mass spectrometry analysis. Four hundred and fifty-seven non-redundant membrane proteins were identified, of which 23% (105) were integral membrane proteins with one or more transmembrane domains. One hundred and fifty-three (33.5%) had no location annotation and 68 were unknown-function proteins. The proteins from different fractions were complementory. A database search for all identified proteins revealed that 53 proteins were involved in the cell communication pathway. More interestingly, more than 50% of the proteins had a protein abundance index concentration of less than 0.1 mol/l, and 12% proteins a concentration 100 times less than that of arginase 1 and actin.
Insights
Researchers identified 457 rat liver membrane proteins, including 105 integral membrane proteins. This study offers methods for human liver proteome projects, focusing on low-abundance proteins.
Area of Science:
- Proteomics
- Cell Biology
- Biochemistry
Background:
- Characterizing membrane proteins is crucial for understanding cellular functions.
- Low-copy integral membrane proteins are particularly challenging to study.
- Developing robust methods is essential for comprehensive human liver proteome projects.
Purpose of the Study:
- To identify and characterize low-copy integral membrane proteins in rat liver plasma membranes.
- To establish effective fractionation and analysis methods applicable to human liver proteome initiatives.
- To provide insights into the abundance and function of identified membrane proteins.
Main Methods:
- Rat liver plasma membrane (PM) fractionation using sucrose density gradient centrifugation.
- Chemical treatments including Na(2)CO(3), chloroform/methanol, and Triton X-100.
- Protein separation by electrophoresis followed by mass spectrometry analysis.
Main Results:
- Identification of 457 non-redundant membrane proteins, with 23% (105) classified as integral membrane proteins.
- Discovery of 153 proteins lacking location annotation and 68 with unknown functions.
- Quantification revealing over 50% of proteins with abundance index < 0.1 mol/L, and 12% at significantly lower concentrations than reference proteins.
Conclusions:
- The study successfully characterized a significant number of integral membrane proteins, including low-abundance ones.
- The developed methodology provides a valuable framework for future human liver proteome research.
- The identified proteins offer potential targets for understanding cell communication and liver function.
