Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes.
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.
Chang
Ing-Feng
IF
Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA. ifchang@unr.edu
D000345:Affinity Labels / Q000737:chemistry; D000939:Epitopes / Q000032:analysis*; D005057:Eukaryotic Cells / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D046912:Multiprotein Complexes / Q000032:analysis* / Q000276:immunology; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000345:Affinity Labels; D000939:Epitopes; D046912:Multiprotein Complexes; D011506:Proteins