Optimization and ZSPORE Analysis of Affinity Purification Coupled with Tandem Mass Spectrometry in Mammalian Cells
Journal Title: Journal of Proteomics and Genomics Research - Year 2012, Vol 1, Issue 1
Abstract
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.
Authors and Affiliations
Shitao Li, Martin E. Dorf
Keywords
Related Articles
- EP ID EP261057
- DOI 10.14302/issn.2326-0793.jpgr-12-100
- Views 124
- Downloads 0
Carbofuran Abrogates the Membrane Ca++-Atpase Activity in Liver: The Role of Cholesterol
Carbofuran is a broad spectrum pesticide used in agricultural fields and domestic places throughout the world. It is one of the deadly toxic carbamate pesticide that kills the pest by inhibiting the crucial enzyme of ner...
Eukaryotic Signature Proteins
Eukaryotic Signature Proteins (ESPs) are proteins that delineate the eukaryotes from the archaea and bacteria. They have no recognisable homologues in any prokaryotic genome, but their homologues are present in all main...
Proteome and Proteomics: from Single Protein to Whole Body
No Abstract
Editorial for Journal of Proteomics and Genomics Research
No Abstract
Determination of the Proteomic Response to Lapatinib Treatment using a Comprehensive and Reproducible Ion-Current-Based Proteomics Strategy
Lapatinib, a small molecule tyrosine kinase inhibitor is currently used in the treatment of HER2-positive breast cancer. The aim of this study was to further understanding of lapatinib response for the development of nov...