THE DEVELOPMENT OF SAMPLE PREPARATION METHODS INVOLVING DETERGENTS FOR ANALYSIS OF MEMBRANE PROTEINS BY MASS SPECTROMETRY

Abstract

Appropriate solubilization of membrane proteins can be achieved by incorporating detergents such as sodium dodecyl sulfate (SDS). Unfortunately, SDS interferes with liquid chromatography (LC) and mass spectrometry (MS). This thesis presents “MS-friendly’ alternatives to solubilize membrane proteins and provides an evaluation of current protocols for SDS removal. Considering the limitation of SDS in a proteome analysis workflow, fluorinated surfactants have previously been proposed. Our results showed that APFO similar proteome solubilization to that of SDS. Unfortunately, APFO was only marginally more tolerable to LC and MS than SDS. Nonetheless, an important advantage of using APFO is that it can be easily removed from the sample by evaporation. The efficiency of precipitation protocols was explored. Our results demonstrate that high recovery is possible. Quantitative re-solubilization of membrane proteins following precipitation was made possible through addition of 80% formic acid. This solvent system may present a promising pathway for top-down MS analysis.