Document Detail


Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes.
MedLine Citation:
PMID:  17072909     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In recent years, MS has been widely used to study protein complex in eukaryotes. The identification of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultracentrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively high. Alternatively, by use of one- or two-step (tandem affinity purification) epitope-tag affinity purification, protein complexes can be isolated by affinity or immunoaffinity columns. These epitope-tags include protein A, hexahistidine (His), c-Myc, hemaglutinin (HA), calmodulin-binding protein, FLAG, maltose-binding protein, Strep, etc. The isolated protein complex can then be subjected to protease (i.e., trypsin) digestion followed by an MS analysis for protein identification. An example, the epitope-tag purification of the Arabidopsis cytosolic ribosomes, is addressed in this article to show the success of the application. Several representative protein complexes in eukaryotes been isolated and characterized by use of this approach are listed. In this review, the comparison among different tag systems, validation of interacting relationship, and choices of MS analysis method are addressed. The successful rate, advantages, limitations, and challenges of the epitope-tag purification are also discussed.
Authors:
Ing-Feng Chang
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Publication Detail:
Type:  Journal Article; Validation Studies    
Journal Detail:
Title:  Proteomics     Volume:  6     ISSN:  1615-9853     ISO Abbreviation:  Proteomics     Publication Date:  2006 Dec 
Date Detail:
Created Date:  2006-12-04     Completed Date:  2007-02-06     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101092707     Medline TA:  Proteomics     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  6158-66     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA. ifchang@unr.edu
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MeSH Terms
Descriptor/Qualifier:
Affinity Labels / chemistry
Epitopes / analysis*
Eukaryotic Cells / metabolism
Gene Expression Profiling / methods
Multiprotein Complexes / analysis*,  immunology
Protein Interaction Mapping / methods
Proteins / immunology,  isolation & purification
Proteomics / methods*
Tandem Mass Spectrometry / methods*
Chemical
Reg. No./Substance:
0/Affinity Labels; 0/Epitopes; 0/Multiprotein Complexes; 0/Proteins

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