**2.6.11 2-DE protein pattern in classification of carcinoma cells**

High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful research tool for the analytical separation of cellular proteins. The qualitative and quantitative pattern of polypeptides synthesized by a cell represents its phenotype and thus defines characteristics such as the morphology and the biological behavior of the cell. By analyzing and comparing the protein patterns of different cells, it is possible to recognize the cell type and to identify the most typical features of these cells. In applied pathology it is

Two Dimensional Gel Electrophoresis in Cancer Proteomics 387

important tool, especially for clinical laboratories involved in the determination of protein expression levels and disease biomarker discovery. When absolute biological variation between samples is the main objective, as in biomarker discovery, 2D-DIGE is the method of choice. While there has been significant progress in nongel (or solution-based) methods for coupling fractionation methods directly online with MS analysis, 2DPAGE has remained a popular technique for conducting proteomic studies. Though 2D-PAGE, like any fractionation scheme, has its advantages and disadvantages, there is no doubt that it will remain an essential technique for the characterization of proteomes for many years to come.

Proteome analysis is a direct measurement of proteins in terms of their presence and relative abundance (Wilkins *et al.,* 1996). The overall aim of a proteomic study is characterization of the complex network of cell regulation. Neither the genomic DNA code of an organism nor the amount of mRNA that is expressed for each gene product (protein) yields an accurate picture of the state of a living cell (Lubec *et al.,* 1999), which can be altered by many conditions. Proteome analysis is required to determine which proteins have been conditionally expressed, how strongly, and whether any posttranslational modifications are affected. Two or more different states of a cell or an organism (e.g., healthy and diseased tissue) can be compared and an attempt made to identify specific qualitative and quantitative protein changes. One of the greatest challenges of proteome analysis is the reproducible fractionation of these complex protein mixtures while retaining the qualitative and quantitative relationships. Currently, twodimensional polyacrylamide gel electrophoresis (2-D PAGE) is the only method that can handle this task (Cutler et al., 1999, Fegatella et al., 1999, Görg et al., 2000), and hence has gained special importance. Since 2-D PAGE is capable of resolving over 1,800 proteins in a single gel (Choe & Lee, 2000), it is important as the primary tool of proteomics research where multiple proteins must be separated for parallel analysis. It allows hundreds to thousands of gene products to be analyzed simultaneously. In combination with computer assisted image evaluation systems for comprehensive qualitative and quantitative examination of proteomes, this electrophoresis technique allows cataloging and comparison of data among groups of

Two-dimensional gel electrophoresis and MALDI-MS are an effective strategy for determining the protein domains present in those gel spots that are observed at significantly lower MW values than are given in the database. While average sequence coverage is only 30%, the peptides detected are confined to a specific region of the protein, such as the protein N- or Cterminal. This information could easily be incorporated into protein identification tables. Regional coverage information is not readily available from either LC-MS/MS analysis of digests of cellular lysates or from epitope-specific antibodies. Some of the protein fragments correspond to chains produced by known cellular processing and activation pathways. Others have been detected as functional and structural domains during in vitro experiments or noted in other in vivo studies, indicating they function intra- or extra-cellularly. By using tools that allow both protein identification and measurement of MW, we can assess the abundance and distribution of protein fragments. Correlation of these results with targeted functional studies

on specific proteins will elucidate the biological function of protein fragments.

**2.6.14 Two-dimensional electrophoresis for cancer proteomics** 

researchers.

**3. Conclusion** 

often difficult to identify the tissue of origin and the stage or grade of a neoplasia by cellular morphology analyzed by classical or immunostaining procedures. The protein pattern itself is the most characteristic feature of a cell and should therefore contribute to the identification of the cell type. For this reason, we separated protein fractions originating from different lung tumor cell lines using 2-D PAGE and we compared the resulting patterns on a multivariate statistical level using correspondence analysis (CA) and ascendant hierarchical clustering (AHC). The results indicate that (i) protein patterns are highly typical for cells and that (ii) the comparison of the protein patterns of a set of interesting cell types allows the identification of potentially new marker proteins. 2-D PAGE is thus a unique and powerful tool for molecular cytology or histopathology, unveiling the protein expression level of tissues or cells.

### **2.6.12 2-DE in understanding Ovarian intratumoral heterogeneity**

The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Twodimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. The tumor cells have been prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression is low. In contrast, large differences have been observed when the protein profiles of different tumors are compared. The differences in gene expression between pairs of malignant carcinomas are slightly larger than the differences observed between pairs of benign tumors. Hence, the 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.

### **2.6.13 Strengths and weaknesses of 2D-PAGE**

Electrophoresis is an established technique that has undergone several advances that have enhanced resolution, detection, quantization, and reproducibility. The 2-D SDS-PAGE and 2DDIGE approaches to protein profiling are accessible and economical methods that possess high resolving power and enable the detection of hundreds of proteins on a single gel plate. Although reproducibility has been an issue with 2D-PAGE, especially when profiling two protein mixtures, it has been greatly improved with the use of 2D-DIGE. Resolution has been enhanced by the introduction of IPGs, which enable the analyst to tailor the pH gradient for maximum resolution using ultrazoom gels with a narrow pH gradient range. With modern 2D-PAGE, it is not unusual to resolve two proteins that differ in pI by 0.001 U. Although 2D-PAGE has been limited by its inability to resolve proteins that are too basic or too acidic, too large or too small, this limitation is continuously diminishing. For example, the separation of basic proteins can be analyzed using IPGs in the pH range of 4–12. Separation science is always evolving, and it will not be long before the remaining issues of gel electrophoresis are adequately resolved. The introduction of 2D-DIGE contributed immensely to solving problems of reproducibility and quantitation. The use of imagers and computers allows not only fast data mining, acquisition, and analysis but also spot detection, normalization, protein profiling, background correction, and reporting and exporting of data. As a separation, detection, and quantitation technique, 2D-DIGE is an

often difficult to identify the tissue of origin and the stage or grade of a neoplasia by cellular morphology analyzed by classical or immunostaining procedures. The protein pattern itself is the most characteristic feature of a cell and should therefore contribute to the identification of the cell type. For this reason, we separated protein fractions originating from different lung tumor cell lines using 2-D PAGE and we compared the resulting patterns on a multivariate statistical level using correspondence analysis (CA) and ascendant hierarchical clustering (AHC). The results indicate that (i) protein patterns are highly typical for cells and that (ii) the comparison of the protein patterns of a set of interesting cell types allows the identification of potentially new marker proteins. 2-D PAGE is thus a unique and powerful tool for molecular cytology or histopathology, unveiling the

The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Twodimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. The tumor cells have been prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression is low. In contrast, large differences have been observed when the protein profiles of different tumors are compared. The differences in gene expression between pairs of malignant carcinomas are slightly larger than the differences observed between pairs of benign tumors. Hence, the 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.

Electrophoresis is an established technique that has undergone several advances that have enhanced resolution, detection, quantization, and reproducibility. The 2-D SDS-PAGE and 2DDIGE approaches to protein profiling are accessible and economical methods that possess high resolving power and enable the detection of hundreds of proteins on a single gel plate. Although reproducibility has been an issue with 2D-PAGE, especially when profiling two protein mixtures, it has been greatly improved with the use of 2D-DIGE. Resolution has been enhanced by the introduction of IPGs, which enable the analyst to tailor the pH gradient for maximum resolution using ultrazoom gels with a narrow pH gradient range. With modern 2D-PAGE, it is not unusual to resolve two proteins that differ in pI by 0.001 U. Although 2D-PAGE has been limited by its inability to resolve proteins that are too basic or too acidic, too large or too small, this limitation is continuously diminishing. For example, the separation of basic proteins can be analyzed using IPGs in the pH range of 4–12. Separation science is always evolving, and it will not be long before the remaining issues of gel electrophoresis are adequately resolved. The introduction of 2D-DIGE contributed immensely to solving problems of reproducibility and quantitation. The use of imagers and computers allows not only fast data mining, acquisition, and analysis but also spot detection, normalization, protein profiling, background correction, and reporting and exporting of data. As a separation, detection, and quantitation technique, 2D-DIGE is an

protein expression level of tissues or cells.

**2.6.13 Strengths and weaknesses of 2D-PAGE** 

**2.6.12 2-DE in understanding Ovarian intratumoral heterogeneity** 

important tool, especially for clinical laboratories involved in the determination of protein expression levels and disease biomarker discovery. When absolute biological variation between samples is the main objective, as in biomarker discovery, 2D-DIGE is the method of choice. While there has been significant progress in nongel (or solution-based) methods for coupling fractionation methods directly online with MS analysis, 2DPAGE has remained a popular technique for conducting proteomic studies. Though 2D-PAGE, like any fractionation scheme, has its advantages and disadvantages, there is no doubt that it will remain an essential technique for the characterization of proteomes for many years to come.

#### **2.6.14 Two-dimensional electrophoresis for cancer proteomics**

Proteome analysis is a direct measurement of proteins in terms of their presence and relative abundance (Wilkins *et al.,* 1996). The overall aim of a proteomic study is characterization of the complex network of cell regulation. Neither the genomic DNA code of an organism nor the amount of mRNA that is expressed for each gene product (protein) yields an accurate picture of the state of a living cell (Lubec *et al.,* 1999), which can be altered by many conditions. Proteome analysis is required to determine which proteins have been conditionally expressed, how strongly, and whether any posttranslational modifications are affected. Two or more different states of a cell or an organism (e.g., healthy and diseased tissue) can be compared and an attempt made to identify specific qualitative and quantitative protein changes. One of the greatest challenges of proteome analysis is the reproducible fractionation of these complex protein mixtures while retaining the qualitative and quantitative relationships. Currently, twodimensional polyacrylamide gel electrophoresis (2-D PAGE) is the only method that can handle this task (Cutler et al., 1999, Fegatella et al., 1999, Görg et al., 2000), and hence has gained special importance. Since 2-D PAGE is capable of resolving over 1,800 proteins in a single gel (Choe & Lee, 2000), it is important as the primary tool of proteomics research where multiple proteins must be separated for parallel analysis. It allows hundreds to thousands of gene products to be analyzed simultaneously. In combination with computer assisted image evaluation systems for comprehensive qualitative and quantitative examination of proteomes, this electrophoresis technique allows cataloging and comparison of data among groups of researchers.
