Recovery and immunoaffinity enrichment of integral membrane proteins from metastatic ovarian cancer tissue

摘要

Integral membrane proteins play key biological roles in cell signaling, transport and 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 intracellular and extracellular domains from the generally insoluble transmembrane regions. Such peptidization separates the intracellular, extracellular and transmembrane components of membrane proteins, resulting in significant informational loss. Here we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including G-protein-coupled receptors) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new buffer system, the ProteoSolve?-TD buffers system is fully compatible with immunoaffinity methods (e.g., enzyme-linked immunosorbent assay and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots and mass spectrometry).