In vivo profiling endogenous interactions with knock-out in mammalian cells.
To precisely identify and screen target-specific protein-protein interactions at the endogenous level, here we introduce a novel quantitative proteomic method we have termed in vivo Profiling Endogenous Interactions with Knock-out (iPEIK). In our design, mouse embryonic fibroblasts (MEFs) derived from target gene knockout (KO) mice can be stable isotope-tagged and serve as a target-free background to "light-up" the target protein-specific protein complex formed in the corresponding wild-type (WT) cells. In mass spectrometric analysis of the pairs of non-labeled versus heavy isotope-labeled peptide signals derived from WT versus KO cells, respectively, we then quantitatively measured the abundance differences of the proteins in the complex immunoprecipitated (IP) from the target-expressing WT versus target-absent KO cells, respectively. Those proteins detected with little or no presence in the cells of KO origin were determined as target-specific interacting partners. Further, dynamic interactors could be identified through different IP mixing schemes. Using iPEIK we identified multiple interacting partners both previously known and unknown to be associated with mitogen-activated protein kinase kinase kinase 2 (MEKK2). Because of the availability of a large library of knockout mice models with various target proteins of biological interests our method is generally applicable to screen any endogenous target-specific PPIs of physiological relevance.
Xie
Ling
L
Department of Biochemistry & Biophysics, School of Medicine, University of North Carolina, 120 Mason Farm Road, Genetic Medicine, Ste 3010, Campus Box No. 7260, Chapel Hill, North Carolina 27599-7260, USA.
Jing
Linhong
L
Yu
Yanbao
Y
Nakamura
Kazuhiro
K
Parker
Carol E
CE
Johnson
Gary L
GL
Chen
Xian
X
D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D047468:Immunoprecipitation; D048730:MAP Kinase Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*
D011506:Proteins; D048730:MAP Kinase Kinase Kinase 2; C482619:Map3k2 protein, mouse