Four-dimensional orthogonal electrophoresis system for screening protein complexes and protein-protein interactions combined with mass spectrometry.
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.
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ABSTRACT: The components that enable cells and organisms to fulfill a plethora of chemical and physical reactions, including their ability to metabolize, replicate, repair and communicate with their environment are mostly based on the functioning of highly complex cellular machines which are to a large extent composed of proteins. With the development of MS techniques compatible with the analysis of minute amounts of biological material, it has become more and more feasible to dissect the composition and modification of these protein machineries. Indeed, new purification methods of protein complexes followed by MS analysis together with the genomic sequencing of various organisms - and in particular of crop species - now provide unforeseen insight to understand biological processes at a molecular level. We here review the current state of the art of in vivo protein complex isolation and their MS-based analytical characterization, emphasizing on the tandem affinity purification approach.Proteomics 11/2010; 11(9):1824-33. · 4.43 Impact Factor
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ABSTRACT: Protein complexes are a cornerstone of many biological processes and together they form various types of molecular machinery. A broad understanding of these protein complexes is crucial for revealing and building models of protein function and regulation. Pancreatic cancer is a highly lethal disease which is difficult to diagnose at early stage and even more difficult to cure. In this study, we applied a gradient clear native gel system combined with subsequent second-dimensional SDS-PAGE to separate protein complexes from cell lysates of SW1990 and PANC-1 pancreatic cancer cell lines with different degrees of differentiation. Ten heat-shock-protein- (HSP-) associated protein complexes were separated and identified, and the differentially expressed proteins related to cancers were also found, such as HSP60, protein disulfide-isomerase A4 (ERp72), and transitional endoplasmic reticulum ATPase (TER ATPase).BioMed Research International 01/2011; 2011:193052. · 2.88 Impact Factor
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ABSTRACT: The aim of this study was to determine the level of students’ environmental knowledge in the Republic of Macedonia. We used two instruments of the random sample of 972 students from 19 secondary schools. The research was conducted in the period 2002-2009. The research has shown that in the research period there is a downward trend of the students’ level of environmental knowledge. Average level of environmental knowledge is 39.19% of the answers right in 2002, and 34.71% in 2009. Students’ environmental knowledge is usually on the level of recognition. © 2011 Published by Elsevier Ltd.Procedia - Social and Behavioral Sciences 01/2011; 15:1395-1400.