Integral membrane protein extraction and biomarker analysis from solid metastatic ovarian tumors

Integral membrane proteins play key biological roles in cell signaling, transport, as well as pathogen invasion. However, quantitative clinical assays for this critical class of proteins (e.g., immunosorbant assays) remain elusive, and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis involve digestion of the generally soluble intra- and extra-cellular domains from the generally-insoluble transmembrane regions. Such peptidization separates the intracellular, extracellular, and transmembrane components of membrane proteins, resulting in significant informational loss. In this paper we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new buffer system. The TD buffer system is fully compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g, 2-D gels, Western blots, and mass spectrometry). We demonstrate near quantitative recovery of the membrane proteins EDG2, EDG4, FAS, KDR, and LAMP-3 by Western blots. Both cytosolic and integral membrane proteins are recovered together in this buffer which is demonstrated in 2-D gels, allowing a more complete picture of the cellular proteome. We also demonstrate how to adapt existing commercial ELISAs for serum-soluble fragments (e.g., sVEGFR2) to measure the tissue titers of the intact membrane proteins from which they originate. Using immunoaffinity enrichment / mass spectrometry we also demonstrate mass spectrometric characterization of proteins recovered from solid tumors with this new method for biomarker validation and isoform characterization.

• Integral membrane protein extraction poster--(5.4MB)