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Yoshigi, M., Pronovost, S.M., Kadrmas, J.L. (2013). Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes.  Proteome Sci. 11(1): 21.
FlyBase ID
FBrf0221867
Publication Type
Research paper
Abstract

Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners-Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5-but has been postulated to bind additional proteins as well.To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry.The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments.

PubMed ID
PubMed Central ID
PMC3688448 (PMC) (EuropePMC)
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    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Proteome Sci.
    Title
    Proteome Science
    Publication Year
    2003-
    ISBN/ISSN
    1477-5956
    Data From Reference
    Alleles (4)
    Genes (38)
    Physical Interactions (8)
    Cell Lines (1)
    Natural transposons (1)
    Experimental Tools (1)
    Transgenic Constructs (2)