2-Dimensional gel electrophoresis

H. Chassaigne, C. C. Chery, G. Bordin, F. Vanhaecke, A. R. Rodriguez, 2-Dimensional gel electrophoresis technique for yeast selenium-containing proteins - sample preparation and MS approaches for processing 2-D gel protein spots, J. Anal. Atom. Spec-trom., 19 (2004), 85-95. 

Hochstrasser, A.-C., James, R. W., Pometta, D., and Hochstrasser, D. (1991). Preparative isoelectrofocusing and high resolution 2-dimensional gel electrophoresis for concentration and purification of proteins. Appl. Theor. Electrophor. 1, 333-337. 

C.S. Giometti, S.L. Tollaksen, C. Chubb, C. Williams and E. Huberman, Analysis of proteins from human breast epithelial-cells using 2-dimensional gel-electrophoresis. Electrophoresis, 16, 1215-1224 (1995). 

Cash, P., Argo, E., and Bruce, K.D. 1995, Characterisation of Haemophilus influenzae proteins by 2-dimensional gel electrophoresis. Electrophoresis 16 135-148. 

Proteins are usually detected by immunohistochemical procedures. Some proteins can be detected directly (e.g. by their errzymatic activity). However, the number of detected proteins depends on the availability of specific antibodies and often determined by the quality and the quantity of the surgical specimen. Immunohistochemical procedures discussed in the prior section have the advantage that the morphology of the tumor is still conserved and can be studied simultaneously with additional information regarding cellular antigens and their localization. In addition to immunohistochemistry, modern proteomics offers new approaches to detect proteins by western blotting, 2-dimensional gel electrophoresis or by protein arrays. 

Wu, R., Sun, Z., Wu, J., Meng, H. and Zhang, H. (2010) Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis. J Dairy Sci 93, 3858-3868. 

Figure 2.1. Schematic illustration oftwo-dimensional gel electrophoresis. Proteins are extracted from the organism of interest and solubilized. The first dimension separates proteins based on isoelectric point. The pi strip is reduced and alkylated and applied to an SDS-PAGE gel for separation by molecular weight. Proteins canbe visualized using a number of staining techniques.

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... 

Rabilloud T (2000) Proteome research two-dimensional gel electrophoresis and identification methods. Springer, Berlin Heidelberg New York... 

The term proteome means the total protein complement of a genome, and pro-teomics means the analysis for proteome. The combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) is a proteomic method of high-throughput analysis of protein expression. By using this 2-DE and MS, proteomic studies have identified many proteins that may be involved in the pathogenic mechanism of cancers. These studies analyzed cancer cell lines, as well as cancer tissues or serum from patients. 

Two-Dimensional Gel Electrophoresis As shown in Figure 3-22, two-dimensional gel electrophoresis allows the separation and display of up to 1,000 different proteins on a single gel. Mass spectrometry (see Box 3-2) can then be used to partially sequence individual protein spots and assign each to a gene. The appearance and nonappearance (or disappearance) of particular protein spots in samples from different tissues, from similar tissues at different stages of development, or from tissues treated in ways that simulate a variety of biological conditions can help define cellular function. 

Rabi I loud, T. (2002) Two-dimensional gel electrophoresis in proteomics old, old fashioned, but it still climbs up the mountains. Proteomics 2, 3-1 0. 

Melle, C., Ernst, G., Schimmel, B., Bleul, A, Koscielny, S., Wiesner, A., Bogumil, R., Moller, U., Osterloh, D., Halbhuber, K.J., and F. Von Eggling, 2003, Biomarker Discovery and Identification in Laser Microdissected Head and Neck Squamous Cell Carcinoma with ProteinChip(R) Technology, Two-dimensional Gel Electrophoresis, Tandem Mass Spectrometry, and Immunohistochemistry. Mol Cell Proteomics. 2(7) 443-52. 

---