Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.
Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.
Zhao
Yingxin
Y
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038, USA.
Zhang
Wei
W
White
Michael A
MA
Zhao
Yingming
Y
D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry
D008565:Membrane Proteins; D020559:Monomeric GTP-Binding Proteins