2D-PAGE analysis

Cerfain proteins were confirmed by Western blot analysis. The 2D PAGE analysis can resolve up to 2000 proteins. In this study, 820 spots were mapped to proteins but only 165 were used in the protein-to-gene analysis. Presumably these represent the highest fold expressed genes from the Ixmg tumors that were also visible on the gels by silver staining. 

Analyzing complex protein patterns by 2D gel electrophoresis has been a research tool since the early 1970s51,68. With this method it is possible to separate and visualize over 1000 distinct proteins in one experiment. Proteins are separated in the first dimension by isoelectric focusing (in a gel that separates proteins based on their relative amounts of acidic and basic amino acids) and in the second dimension by size. The proteins are visualized by staining and then quantified by densitometry. Figure 6 contains an example of a silver-stained 2D-PAGE analysis of liver proteins obtained from control and E2-treated largemouth bass. By visual inspection it is clear that there are numerous proteins expressed in the treated sample that are absent in the control, and there are proteins in the control that are not present in the treated sample. These spots would all be candidates for protein identification. 

A novel technique, laser capture microdissection (LCM), has been developed to obtain very small populations of well-defined normal epithelial and adjacent tumor cells for subsequent lysing. In contrast to the —50,000 LCM-procured cells needed for 2D-PAGE analysis, the SELDl process requires only 25-100 cells to obtain a usable protein profile [47,48]. The technique is also applicable to subsequent imaging analysis [49]. 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

FIGURE 4.12 Experimental 2D-PAGE map of a neuroblastoma xenograft implanted in mice. The circles indicate the spots identified by MS analysis. Reproduced from Campostrini et al. (2005) with permission from Wiley-VCH Verlag GmbH. 

Dondi, F., Bassi, A., Cavazzini, A., Pietrogrande, M.C. (1998). A quantitative theory of the statistical degree of peak overlapping in chromatography. Anal. Chem. 70, 766. Eckerskom, C., Strupat, K., Schleuder, D., Hochstrasser, D.F., Sanchez, J.-C., Lottspeich, F., Hillenkamp, F. (1997). Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting. Anal. Chem. 69, 2888. Expasy,http //www.expasy.ch. 

Fig. 3A, B Strategies for the analysis of complex protein mixtures. A Proteins are separated by 2D PAGE and the relevant protein spots excised from the gel, digested, and analyzed by mass spectrometry. B Proteins are first digested into small peptides and then fractionated by two rounds of chromatography before each peptide is analyzed by mass spectrometry...

Although almost 30 years old, the most widely used technology in comparative proteomics is still 2D PAGE. This applies to the global analysis of proteins... 

However, analysis of protein mixtures derived from cells, tissues, and body fluids by 2D PAGE by no means represents a comprehensive picture of the proteins in the mixture. Proteins with extreme isoelectric points, large proteins, small proteins, and hydrophobic proteins are commonly not amenable to 2D PAGE and hence can be easily missed. Furthermore, low abundant proteins are often not detected in 2D gels when proteins of high abundance are present. This limitation is particularly relevant when analyzing serum or other body fluids, where protein amounts vary by ten orders of magnitude (Anderson and Anderson 1998). 

Using 2D-PAGE, cell cycle entry has been shown to be associated with a significant increase in p27(kipl) phosphorylation in human primary B lymphocytes. Detailed analysis revealed that different cyclins and cyclin-dependent kinases interact with distinct posttranslationally modified isoforms of p27(kipl) in vivo. These results have to be interpretated in the context of overexpression of cyclin D3 in the presence of high levels of p27(kipl) in human B-cell lymphomas with adverse clinical outcome [110]. 

The interest of the consortium in developing and testing new tools for proteome analysis was directed to solutions for particular technical problems concerning sample preparation, the 2D PAGE system, protein quantification, and the development of UniClone sets (nonredundant cDNA expression library) from the adult human brain to be used for creating clinically relevant biochips. These... 

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