Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry
Identifying the partners of a given protein (the interactome) may provide leads about the protein’s function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-i
- PDF / 307,985 Bytes
- 12 Pages / 504.57 x 720 pts Page_size
- 82 Downloads / 251 Views
1
Introduction The interactome may be defined as the complete set of molecular interactions of a molecule in a given cell or other biological environment [1]. The interactome of a protein represents the proteins and molecules that interact with it to promote, regulate, and inhibit its activity or expression. Alternatively, members of the interactome may be substrates or molecular components regulated by the protein of interest. Identifying the interactome of a given protein may provide leads about the protein’s function and the molecular mechanisms in which it is involved. The protein interaction map of Drosophila melanogaster encompasses 556 protein complexes and can assign functional
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_19, © Springer Science+Business Media LLC 2017
223
224
Giuseppina Maccarrone et al.
processes to almost 600 protein-coding genes, many of which previously lacked annotation. The data gained from the Drosophila melanogaster interactome may also provide information about human cell interactomes [2] and enrich genome databases by generating annotations to non-annotated protein-coding genes. An extensive proteomic survey that used affinity tag purification of E. coli strains identified 5,993 protein interactions and allowed proteins to be assigned functions such as protein synthesis, amino acid metabolism, biofilm formation, and motility [3]. More specific to a protein, novel partner proteins in brain tissue were unraveled to collapsin response mediator protein-2 (CRMP2/DPYSL2) (Reference: Martins-deSouza D, Cassoli JS, Nascimento JM, Hensley K, Guest PC, PinzonVelasco AM, Turck CW. The protein interactome of collapsin response mediator protein-2 (CRMP2/DPYSL2) reveals novel partner proteins in brain tissue. Proteomics Clin Appl. 2015 Oct;9(9–10):817–31). The study of protein interactomes in diseases may reveal dysfunctional pathways, further insights into regulation, and the possible role that protein partners play in the disease [4]. Many interactome studies have been performed with yeast two-hybrid screening (Y2H) and Tandem Affinity Purification (TAP) [5], although co-immunoprecipitation (co-IP) is also a reliable method for studying protein interactomes with its own advantages [6]. Briefly, the co-IP of intact protein complexes employs an immobilized antibody that targets a specific protein. The sample of interest is passed through or incubated with the antibody under non-denaturating conditions. Next, the target protein (bait) binds to the antibody along with its partners (prey), while noninteracting proteins pass through or do not bind to the matrix. The captured protein complex may be analyzed further by proteomic analysis (Fig. 1). Co-IP and subsequent mass spectrometry (MS) have been applied to proteins of interest in diseases. Recently, the B-Raf interactome was characterized in mouse hippocampal cells [7, 8]. This protein is involved in a cellular signaling cascade, known t
Data Loading...
