Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry.
The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.
Vasilescu
Julian
J
The Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Guo
Xuecui
X
Kast
Juergen
J
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002845:Chromatography; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D005557:Formaldehyde / Q000494:pharmacology; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins
D003432:Cross-Linking Reagents; D000939:Epitopes; D011506:Proteins; D005557:Formaldehyde; C109418:Mras protein, mouse; D020559:Monomeric GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins