High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

AbstractWe have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography–mass spectrometry (LC–MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2–5μg of protein. The method is also compatible with our standard LC–MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment