Protein Characterization of Triton Insoluble Detergent Resistant Membrane Fractions in Bovine Rod Outer Segments

Abstract

Abstract: : Purpose: To identify proteins associated with detergent resistant membranes (DRM) prepared from bovine rod outer segments (ROS). Methods: DRM fractions were prepared from bovine ROS membranes by sucrose density gradient centrifugation of Triton X–100 solubilized material. Matrix–assisted laser desorption/ionization time–of–flight mass spectrometer (MALDI–TOF–MS) was performed on proteins separated by one–dimensional SDS–PAGE to identify potential DRM–associated proteins. The identity of the proteins in DRM fractions was confirmed by Western blotting. Results: MALDI–TOF–MS analysis of DRM fractions identified a number of candidate proteins, several of which are important proteins associated with rod structure and phototransduction. Many of the previously reported proteins known to associate with the DRM were identified by MALDI–TOF–MS and confirmed on Western blots from three different perpetrations. Previously unreported proteins including CNG–1, sodium/potassium exchanger, and myosin have been identified by MALDI–TOF–MS as possible candidate proteins, but have not yet been confirmed by Western blot. Conclusions: The proteins associating with the DRM fractions have as yet undetermined properties that increase their affinities for the highly–ordered lipid environment of DRM. Identification of the DRM proteome will provide important information about how lipid domains potentially regulate phototransduction and maintenance of ROS structure.

Keywords: proteomics • lipids