Improving solubilization and digestion techniques for membrane proteomics (2014)

Undergraduates: Stephanie Hess, Stephanie Moore

Faculty Advisor: James Jorgenson
Department: Chemistry

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Membrane proteins are molecules existing within and/or anchored to biological membranes, such as that of a cell or organelle. These proteins act as cellular receptors and transporters, making them particularly significant to the medical and pharmaceutical fields. However, these proteins are difficult to analyze due to their insolubility with traditional solvents and instability outside of a biological membrane environment. Therefore, this research focused on the development of specialized extraction, purification, and digestion techniques required for membrane protein identification. The application of various solvents and detergents were tested to improve solubility, as well as multiple purification techniques to successfully extract and isolate membrane proteins from their native environment. Optimized conditions were selected for each step of the analysis. First intact proteins were separated using high-pressure liquid chromatography and the resulting fractions collected were enzymatically digested. The resulting peptides were then separated via ultra-high pressure liquid chromatography coupled with mass spectrometry and the resulting spectra were used to identify the proteins present. It was found that utilizing sonication throughout the process, along with various solvents and detergents, resulted in an increased number of membrane protein identifications.