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| Most current approaches for purification and identification of protein complexes adopt affinity purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinitypurification. Herein, we propose a new approach, termed as the four-dimensional orthogonal electrophoresis (4-DE) system, to find and analyze the cytoplasmic protein complexes. 4-DE system is composed of two parts: nondenaturing part (Part I) and denaturing part (Part II). Through Part I and decision procedure separations, six protein complex candidates 20S core particle of proteasome (CP), hemoglobin (Hb, α2β2), Hb (α2δ2), peroxiredoxin-2 (PRDX2), carbonic anhydrase-1 (CAH1), and heat shock protein 60 (HSP60) were separated. CP, Hb (α2β2), PRDX2, and HSP60 with different MW's and pI's were chosen for Part II proteomic analysis. The results indicate that 4-DE is not only suitable for studying protein complexes and protein-protein interactions as well as structural proteomics from complex biological samples, but can also be easy to separate and concentrate intact protein complexes from dilute complex samples. |
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| Keywords:Protein complex;Proteomics; Four-dimensional orthogonal electrophoresis;Thin layer IEF;Mass spectrometry;Protein-protein interaction |
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