Two-dimensional gels

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. 

The two-dimensional gel of the yeast extract is shown in Figure 5.18 and clearly illustrates the complexity of the analytical problem. 

Figure 5.18 Silver-stained two-dimensional gel of the proteins extracted from the yeast S. cerevisiae. From Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and KaLkM-nen, N., Rapid Commun. Mass Spectrom., 15, 1685-1692, Copyright 2001. John WUey Sons Limited. Reproduced with permission.

The beauty of 2D gel electrophoresis as a separation technique is the orthogonality of the two separation dimensions separation by charge (isoelectric point) in the first dimension, and separation by size in the second dimension. Two-dimensional gel electropheresis is the core separation technique for proteomics, along with HPLC (for preparative isolation). 

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. 

Henzel, W. J., Billeci, T. M., Stults, J. T., and Wong, S. C. (1993). Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90, 5011-5015. 

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. 

Liu, H. J., Berger, S. J., Chakrahorty, A. B., Plumh, R. S., Cohen, S. A. (2002). Multidimensional chromatography coupled to electrospray ionization time-of-fhght mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J. Chromatogr. B 782(1-2), 267-289. 

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. 

Proteome profiling allows quantitative and global examination of the changes in protein expression in different strains or cells under different conditions. Two-dimensional gel... 

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