Four-Dimensional Orthogonal Electrophoresis System

Four-Dimensional Orthogonal Electrophoresis System for Screening Protein Complexes and Protein−Protein Interactions Combined with Mass Spectrometry

Xiaodong Wang (王曉東), Guoqiang Chen (陳國強), Hui Liu (劉輝), Zhiyun Zhao (趙志云), and Zhili Li* (李智立) *

PMID: 20690703 [PubMed - in process]
J. Proteome Res., 2010, 9 (10), pp 5325–5334
DOI: 10.1021/pr100581x
Publication Date (Web): August 6, 2010

Abstract
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.