Gel electrophoresis , two-dimensional

The readily available experimental tools for measmement of protein expression by two-dimensional electrophoresis (2DE), and for protein identification and characterization by mass spectrometry (MS) have made a significant impact on proteonucs (Hamdan and Righetti, 2(X)5). The coupling of 2DE and MS offers an efficient tool for investigating pro-teome expression. 

The characterization of complex proteomes requires separation, detection and analysis of many thousands of proteins from whole cells, tissues or organisms. Cellular/tissue proteins are solubilized (Herbert, 1999) prior to their separalion/analysis. Two-dimensional gel electrophoresis has dononstrated the potential to separate several thousand of proteins (Klose 1999 RabiUoud, 2(XK)) in single experiment and has been the method of choice for the separation/purificalion in proteomic studies. 

This chapter cannot do justice to the important technique of 2-D PAGE. Consult Refs. 32, 36, 46-50, 59, and 60 and the articles cited therein for details of 2-D PAGE. 

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The Tools of Proteomics A variety of methods and techniques including two-dimensional gel electrophoresis (2DE), capillary liquid chromatography, stable isotope labeling, and mass spectrometry has been developed for qualitative and quantitative protein... 

Two-dimensional gel electrophoresis (2DE) is a two-dimensional technique for protein separation, which combines isoelectric focusing and sodium dodecyl sulphate (SDS) electrophoresis. The high resolving power results from separation according to charge (isoelectric point) in the first dimension and size (mobility in a porous gel) in the second dimension. Depending on the gel size, from several hundred to more than 5,000 proteins can be separated. 

Gygi, S. P., Corthals, G. L., Zhang, Y., Rochon, Y., and Aebersold, R. (2000). Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. USA 97, 9390-9395. 

Celis, J.E., Bravo, R. (1984). Two-Dimensional Gel Electrophoresis of Proteins. Academic Press, New York. 

Rose, D.J., Opiteck, G.J. (1994). Two-dimensional gel electrophoresis/liquid chromatography for the micropreparative isolation of proteins. Anal. Chem. 66(15), 2529-2536. 

Hoffmann, R, Ji, H., Moritz, R. L., Connolly, L. M., Frecklington, D. F., Layton, M. J., Eddes, J. S., Simpson, R. J. (2001). Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215 a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1(7), 807. 

Although classic two-dimensional gel electrophoresis provides exquisite peak capacity, it suffers from several limitations. First, the technology is labor-intensive and difficult to automate, which hampers applications to large-scale proteomics analyses (Hanash, 2000). 

Poehling reported a microscale two-dimensional gel electrophoresis system 25 years ago (Poehling and Neuhogg, 1980 Neuhoff, 2000). In that system, separation in the IEF dimension was performed in thin, gel-filled tubes. After IEF, the gel was transferred to a 3 cm x 3.5 cm polyacrylamide gel, where proteins were separated by SDS-PAGE. Several hundred components were resolved from a few micrograms of protein homogenate. 

Minowa T et al. Proteomic analysis of the small intestine and colon epithelia of adenomatous polyposis coli gene-mutant mice by two-dimensional gel electrophoresis. Electrophoresis 2000 21 1782—1786. 

Edgar PF Comparative proteome analysis. Tissue homogenate from normal human hippocampus subjected to two-dimensional gel electrophoresis and Coo-massie blue protein staining. Mol Psychiatry 2000 5 85-90. 

Unlu M et al. Detection of complement factor B in the cerebrospinal fluid of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy disease using two-dimensional gel electrophoresis and mass spectrometry. Neurosci Lett 2000 282 149-152. 

Weekes J et al. Bovine dilated cardiomyopathy proteomic analysis of an animal model of human dilated cardiomyopathy Electrophoresis 1999 20 898-906. Doherty NS et al. Analysis of changes in acute phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis. Electrophoresis 1998 19 355-363. 

Moller A et al. Two-dimensional gel electrophoresis a powerful method to elucidate cellular responses to toxic compounds. Toxicology 2001 160 129-138. 

Many diseases are characterized by the expression of specific proteins1 in some cases, malignant cells yield unique protein profiles when total cellular protein extracts are analyzed by proteomic methods such as two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS).2 High-throughput proteomic studies may be useful to differentiate normal cells from cancer cells, to identify and define the use of biomarkers for specific cancers, and to characterize the clinical course of disease. Proteomics can also be used to isolate and characterize potential drug targets and to evaluate the efficacy of treatments. 

Table 11.2 Selected genes whose rate of transcription is altered by binding of insulin to its receptor. In virtually all instances, the ultimate effect is to promote anabolic events characteristic of insulin action. Two-dimensional gel electrophoresis has also pinpointed dozens of proteins of unknown function whose cellular level is altered by insulin...

DGE a AC AMS APCI API AP-MALDI APPI ASAP BIRD c CAD CE CF CF-FAB Cl CID cw CZE Da DAPCI DART DC DE DESI DIOS DTIMS EC ECD El ELDI EM ESI ETD eV f FAB FAIMS FD FI FT FTICR two-dimensional gel electrophoresis atto, 10 18 alternating current accelerator mass spectrometry atmospheric pressure chemical ionization atmospheric pressure ionization atmospheric pressure matrix-assisted laser desorption/ionization atmospheric pressure photoionization atmospheric-pressure solids analysis probe blackbody infrared radiative dissociation centi, 10-2 collision-activated dissociation capillary electrophoresis continuous flow continuous flow fast atom bombardment chemical ionization collision-induced dissociation continuous wave capillary zone electrophoresis dalton desorption atmospheric pressure chemical ionization direct analysis in real time direct current delayed extraction desorption electrospray ionization desorption/ionization on silicon drift tube ion mobility spectrometry electrochromatography electron capture dissociation electron ionization electrospray-assisted laser desorption/ionization electron multiplier electrospray ionization electron transfer dissociation electron volt femto, 1CT15 fast atom bombardment field asymmetric waveform ion mobility spectrometry field desorption field ionization Fourier transform Fourier transform ion cyclotron resonance... 

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