Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes.
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 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.
Fonslow
Bryan R
BR
Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, California 92037, USA.
Kang
Seong A
SA
Gestaut
Daniel R
DR
Graczyk
Beth
B
Davis
Trisha N
TN
Sabatini
David M
DM
Yates
John R
JR
D002021:Buffers; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification; D002460:Cell Line; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D000076222:Mechanistic Target of Rapamycin Complex 1; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification; D058570:TOR Serine-Threonine Kinases; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification; D014783:Viscosity
D002021:Buffers; D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; C471213:His-His-His-His-His-His; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010455:Peptides; D020033:Protein Isoforms; D021122:Protein Subunits; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D006639:Histidine; C085706:FLAG peptide; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases