Optimization and Zspore Analysis of Affinity Purification Coupled With Tandem Mass Spectrometry in Mammalian Cells - Optimization and Zspore Analysis of Affinity Purification Coupled With Tandem Mass Spectrometry in Mammalian Cells - Open Access Pub

Defining protein-protein interactions is essential for understanding the mechanisms by which cells regulate basic functions, such as metabolism, transcription, and signal transduction. Affinity purification followed by tandem mass spectrometry (AP-MS) has application for discovery of new interactors regulating various cellular processes. Here we optimize the purification method for AP-MS and develop a simplified unbiased analytical tool, Z-score plus prey occurrence and reproducibility (ZSPORE) for data analysis. Using this pipeline we achieve a higher efficiency of AP-MS and enhanced identification of high confidence interacting proteins (HCIP) in mammalian cells. When applied to analysis of the innate immune interactome, these methods enhanced HCIP identification. In addition, we investigated the GRB2 complex, which is associated with signal transduction and cell growth. Twenty-four known GRB2 interacting proteins were identified plus 26 new GRB2 binding partners. Thus, these straightforward methods recapitulate known protein interactions, discover novel complexes, and allow mapping of protein interaction networks. DOI10.14302/issn.2326-0793.jpgr-12-100 Analysis of protein-protein interaction has contributed numerous insights for understanding the regulation of antiviral defense, DNA repair, autophagy, and immune signaling pathways. Discerning how proteins interact in complex and dynamic networks is a key for dissecting the complexity of many genotype-to-phenotype relationships. Proteomics has emerged as a powerful tool to analyze multicomponent complexes formed under close to physiological conditions. Among various proteomic based methods, affinity purification followed by tandem mass spectrometry (AP-MS) has proven to be highly successful for identification of interacting proteins. Using this approach, global wide interactomes have been established in Escherichia coli 1, Mycoplasma pneumonia 2, Saccharomyces cerevisiae 3, 4, 5, Drosophila melanogaster 6, and HIV-host 7. In vertebrates AP-MS has been used to define proteomic subspaces and specific signal pathways for the antiviral innate immunity pathway 8, autophagy 9, deubiquitinase interactome 10, endoplasmic reticulum-associated protein degradation network 11, tumor necrosis factor signaling 12, proteasome interaction network 13, and disease related protein networks 14. Various affinity tags have been employed for protein purification, but the FLAG and HA epitopes remain the most popular tags for AP-MS in mammalian cells. To optimize the AP-MS method, we compare purification strategies using the