Affinity enrichment of plasma membrane for proteomics analysis.
Proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially a powerful approach for the identification of membrane-associated proteins responding to these environments. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. Here, we describe an affinity enrichment method, which combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads, for the isolation of plasma membranes. This method results in a 400-fold enrichment of plasma membrane relative to endoplasmic reticulum, a major contaminant in standard plasma membrane preparations, and dramatically reduces contamination from other cellular organelles. The biotinylation reaction did not interfere with ligand-dependent activation of receptor tyrosine kinases or G-protein coupled receptors, suggesting cell-surface signal transduction machinery remains functional. Membrane fractions prepared by this method should provide excellent starting materials for membrane proteomics analysis such as studies of dynamic trafficking and regulation of signaling molecules or identification of disease-specific membrane markers.
Zhang
Wei
W
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA.
Zhou
Ge
G
Zhao
Yingxin
Y
White
Michael A
MA
Zhao
Yingming
Y
D019921:Biotinylation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D008863:Microspheres; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D019809:Streptavidin
D008565:Membrane Proteins; D020543:Proteome; D019809:Streptavidin; D020928:Mitogen-Activated Protein Kinases