Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction.

ABSTRACT: While mass spectrometry (MS) plays a key role in proteomics research, characterization of membrane proteins (MP) by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND) at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids

SUBMITTER: Pham MD 

PROVIDER: S-EPMC5503057 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature