Journal
METHODS
Volume 53, Issue 4, Pages 453-459Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2010.12.016
Keywords
QUBIC; Protein-protein interaction; Quantitative mass spectrometry; Proteomics; BAC transgeneomics; Label-free quantification; SILAC
Funding
- German National Genome Research Network
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Large-scale proteomic screens are increasingly employed for placing genes into specific pathways. Therefore generic methods providing a physiological context for protein-protein interaction studies are of great interest. In recent years many protein-protein interactions have been determined by affinity purification followed by mass spectrometry (AP-MS). Among many different AP-MS approaches, the recently developed Quantitative BAC InteraCtomics (QUBIC) approach is particularly attractive as it uses tagged, full-length baits that are expressed under endogenous control. For QUBIC large cell line collections expressing tagged proteins from BAC transgenes or gene trap loci have been developed and are freely available. Here we describe detailed workflows on how to obtain specific protein binding partners with high confidence under physiological conditions. The methods are based on fast, streamlined and generic purification procedures followed by single run liquid chromatography-mass spectrometric analysis. Quantification is achieved either by the stable isotope labeling of amino acids in cell culture (SILAC) method or by a 'label-free' procedure. In either case data analysis is performed by using the freely available MaxQuant environment. The QUBIC approach enables biologists with access to high resolution mass spectrometry to perform small and large-scale protein interactome mappings. (C) 2010 Elsevier Inc. All rights reserved.
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