Anal. Chem. 2010, 82, 6643–6651
Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes Bryan R. Fonslow,† Seong A. Kang,‡ Daniel R. Gestaut,| Beth Graczyk,| Trisha N. Davis,| David M. Sabatini,‡,§ and John R. Yates III*,† Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, California 92037, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Department of Biochemistry, University of Washington, Seattle, Washington 98195 Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 °C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (
