Identification of mammalian protein complexes by lentiviral-based affinity purification and mass spectrometry.
ABSTRACT Protein complexes and protein-protein interactions (PPIs) are fundamental for most biological functions. Deciphering the extensive protein interaction networks that occur within cellular contexts has become a logical extension to the human genome project. Proteome-scale interactome analysis of mammalian systems requires efficient methods for accurately detecting PPIs with specific considerations for the intrinsic technical challenges of mammalian genome manipulation. In this chapter, we outline in detail an innovative lentiviral-based functional proteomic approach that can be used to rapidly characterize protein complexes from a broad range of mammalian cell lines. This method integrates the following key features: (1) lentiviral elements for efficient delivery of tagged constructs into mammalian cell lines; (2) site-specific Gateway™ recombination sites for easy cloning; (3) versatile epitope-tagging system for flexible affinity purification strategies; and (4) LC-MS-based protein identification using tandem mass spectrometry.
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ABSTRACT: Proteins play a fundamental role in establishing the diversity of cellular processes in health or disease systems. This diversity is accomplished by a vast array of protein functions. In fact, a protein rarely has a single function. The majority of proteins are involved in numerous cellular processes, and these multiple functions are made possible by interactions with other molecules. The complexity of interactions is substantially increased by the spatial and temporal diversity of proteins. For example, proteins can be part of distinct complexes within different subcellular compartments or at different stages of the cell cycle. Posttranslational modifications can regulate and further expand the ability of proteins to establish localization- or temporal-dependent interactions. This complexity and functional divergence of interactions is further increased by the simultaneous presence of stable, transient, direct, and indirect protein interactions. Thus, an understanding of protein functions cannot be fully accomplished without knowledge of its interactions. Characterizing these interactions is therefore critical to understanding the biology of health and disease systems.Analytical Chemistry 11/2012; · 5.70 Impact Factor
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ABSTRACT: While major progress has been achieved in the experimental techniques used for the detection of protein interactions and in the processing and analysis of the vast amount of data that they generate, we still do not understand why the set of identified interactions remains so highly dependent on the particular detection method. Here we present an overview of the major high-throughput experimental methods used to detect interactions and the datasets produced using these methods over the last 10 years. We discuss the challenges of assessing the quality of these datasets, and examine key factors that likely underlie the persistent poor overlap between the interactions detected by different methods. Lastly, we present a brief overview of the literature-curated protein interaction data stored in public databases, which are often relied upon for independent validation of newly derived interaction networks.Current Opinion in Structural Biology 09/2013; · 8.74 Impact Factor