High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins
Berridge G., Chalk R., D'Avanzo N., Dong L., Doyle D., Kim JI., Xia X., Burgess-Brown N., Deriso A., Carpenter EP., Gileadi O.
We 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 0.2-5 μg 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. © 2010 Elsevier Inc. All rights reserved.