Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane

Yingxin Zhao, Wei Zhang, Michael A. White, Yingming Zhao

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

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 pg of affinity-purified plasma membrane proteins led to the idenfification 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 (∼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.

Original languageEnglish (US)
Pages (from-to)3751-3757
Number of pages7
JournalAnalytical Chemistry
Volume75
Issue number15
DOIs
StatePublished - Aug 1 2003
Externally publishedYes

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High performance liquid chromatography
Cell membranes
Membrane Proteins
Proteins
Mitochondria
Contamination
Membranes
Molecules
Streptavidin
Epidermal Growth Factor Receptor
Integrins
Purification
Impurities

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane. / Zhao, Yingxin; Zhang, Wei; White, Michael A.; Zhao, Yingming.

In: Analytical Chemistry, Vol. 75, No. 15, 01.08.2003, p. 3751-3757.

Research output: Contribution to journalArticle

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