Four-dimensional orthogonal electrophoresis system for screening protein complexes and protein-protein interactions combined with mass spectrometry.
Most current approaches for purification and identification of protein complexes adopt affinity purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinity purification. 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 MWs 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.
Wang
Xiaodong
X
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China.
Chen
Guoqiang
G
Liu
Hui
H
Zhao
Zhiyun
Z
Li
Zhili
Z
D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D046912:Multiprotein Complexes