**Two Dimensional Gel Electrophoresis in Cancer Proteomics**

Soundarapandian Kannan1, Mohanan V. Sujitha1, Shenbagamoorthy Sundarraj1 and Ramasamy Thirumurugan2 *1Proteomics and Molecular Cell Physiology Lab Department of Zoology, Bharathiar University, Coimbatore 2Department of Animal Science, Bharathidasan University, Tiruchirappalli India* 

#### **1. Introduction**

358 Gel Electrophoresis – Advanced Techniques

Zensi, A.; Begley, D.; Pontikis, C.; Legros, C.; Mihoreanu, L.; Buchel, C. & Kreuter, J. (2010).

No.10, pp. 842–848, ISSN 1029-2330

Human Serum Albumin Nanoparticles Modified with Apolipoprotein a-I Cross the Blood-Brain Barrier and Enter the Rodent Brain. *Journal of Drug Targeting*, Vol.18,

> Two-dimensional electrophoresis (2-DE) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique sort's protein according to two independent properties in two discrete steps: the first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights (Mr, relative molecular weight). Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Thousands of proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein obtained. The separation of proteins by 2-DE dates back to the 1950s. The rst 2-DE technique was developed by Smithies and Poulik in 1956 and O'Farrell, 1975 and Klose, 1975 significantly modified this method to elucidate protein profile. In the original technique, the first-dimension separation was performed in carrier ampholyte-containing polyacrylamide gels cast in narrow tubes.

> The power of 2-DE as a biochemical separation technique has been recognized virtually since its introduction. Its application, however, has become significant only in the last few years because of a number of developments. The introduction of immobilized pH gradients and Immobiline™ reagents brought superior resolution and reproducibility to firstdimension IEF. Based on this concept, Görg *et al*., 1989 and Gorg, 1991 developed the currently employed 2-D technique, where carrier ampholyte-generated pH gradients have been replaced with immobilized pH gradients and tube gels replaced with gels supported by a plastic backing. New mass spectrometry techniques have been developed that allow rapid identification and characterization of very small quantities of peptides and proteins extracted from single 2-D spots. More powerful, less expensive computers and software are now available, rendering thorough computerized evaluations of the highly complex 2-D patterns economically feasible. Data about entire genomes (or substantial fractions thereof) for a number of organisms are now available, allowing rapid identification of the gene encoding a protein separated by 2-DE. The World Wide Web provides simple, direct access

Two Dimensional Gel Electrophoresis in Cancer Proteomics 361

protein interaction, very few can provide detailed structural information. NMR spectroscopy is one of these, and in recent years several complementary NMR approaches, including residual dipolar couplings and the use of paramagnetic effects, have been

Two-dimensional gel electrophoresis for separation of complex protein samples coupled with mass spectrometry for protein identification has been used to analyze protein expression patterns for many sample types. Inherent in the use of this technique is information on not only full-length protein expression, but expression of modified, splice variant, cleavage product, and processed proteins. Any protein modification that leads to a change in overall protein charge and/or molecular weight (MW) will generate a different spot on the 2-DE. Modification specific staining can identify whether a specific posttranslational modification is responsible for the shift, and mass spectrometry can potentially identify the source of isoelectric point (pI) and/or MW differences. Due to the lack of complete coverage for a protein's amino acid sequence using either matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or high-performance liquid chromatography (HPLC) tandem mass spectrometry (LC-MS/MS), there has been limited success in using MS to identify isoforms and post-translational modifications. While the theoretical MW is often slightly higher than the MW of the fully processed protein due to cleavage of signal and pro-peptides, there can also be post-translational modifications that increase the protein's gel MW. Thus, an exploration into the causes of the difference in the theoretical MW and the MW as seen in the gel can yield information about the state of the protein. When the gel MW of a given protein is significantly lower than the calculated weight, the gel spot represents a protein fragment. The extent to which proteins are present as fragments or variants in tissues and fluids has not been determined, but the combination of 2-DE, Western blotting, and mass spectrometry–based protein identification makes such analyses possible. Two-dimensional gel electrophoresis of human mammary tissue, followed by immune blotting, resulted in multiple spots at significantly differing molecular weights. The function of protein fragments is dependent on activation processes and localization properties. This Chapter will be critically analyzed as per the contents given in

Efficient and reproducible sample preparation methods are a key to successful 2-DE (Rabilloud 1999, Macri *et al.* 2000, Molloy 2000). Sample preparation methods range from extraction with simple solubilization solutions to complex mixtures of chaotropic agents, detergents, and reducing agents. Sample preparation can include enrichment strategies for

3. Prevent postextraction chemical modification, including enzymatic or chemical

1. Reproducibly solubilize proteins of all classes, including hydrophobic proteins

2. Prevent protein aggregation and loss of solubility during focusing

developed that can provide insight into the structure of protein–protein complexes.

the synopsis with up-to-date informations.

**2. Overview of experimental design** 

separating protein mixtures into reproducible fractions.

An effective sample preparation procedure will:

degradation of the protein sample

**2.1 Experimental design 2.1.1 Sample preparation**

to spot pattern databases for the comparison of electrophoresis results and genome sequence databases for assignment of sequence information.

A large and growing application of 2-DE in "proteome analysis." Proteome analysis is "the analysis of the entire Protein complement expressed by a genome". The analysis involves the systematic separation, identification, and quantification of many proteins simultaneously from a single sample. Two-dimensional electrophoresis is used e due to its unparalleled ability to separate thousands of proteins simultaneously. Two-dimensional electrophoresis is also unique in its ability to detect post- and co-translational modifications, which cannot be predicted from the genome sequence. Applications of 2-DEinclude proteome analysis, cell differentiation, and detection of disease markers, monitoring therapies, drug discovery, cancer research, purity checks, and microscale protein purification.

"Proteomics" is the large-scale screening of the proteins of a cell, organism or biological fluid, a process, which requires stringently controlled steps of sample preparation, 2-DE, image detection and analysis, spot identification, and database searches. Moreover, Proteomics studies lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, developmental stages etc. Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression. A keystone of conventional proteomics is high-resolution 2D gel electrophoresis followed by protein identification using mass spectrometry.

As a technique with high-flux and high resolution, proteomics has been widely applied in proteome analysis of tumors. The onset and development of the tissues and cells can be detected at the entire protein level through analyzing the differential expression of proteins. The combination of 2-DE and mass spectrometry can be used to identify differential proteins between tumor cells and normal original cells, and these differential proteins imply a large quantity of biological information. Some of the special proteins are special markers of tumors. The most consistently successful proteomic method is the combination of twodimensional gel electrophoresis (2DE) for protein separation, visualization, and mass spectrometric (MS) identification of proteins using peptide mass fingerprints and tandem MS peptide sequencing.

The experiments form the basis of proteomics, and present significant challenges in data analysis, storage and querying. The core technology of proteomics is 2-DE. At present, there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure. The replacement of classical first-dimension carrier ampholyte pH gradients with well-defined immobilized pH gradients has resulted in higher resolution, improved inter-laboratory reproducibility, higher protein loading capacity, and an extended basic pH limit for 2-DE. With the increased protein capacity, micropreparative 2-DE has accelerated spot identification by mass spectrometry and Edman sequencing. The remarkable improvements in 2-DE resulting from immobilized pH gradient gels, together with convenient new instruments for IPG-IEF, will make critical contributions to advances in proteome analysis.

A comprehensive understanding of protein–protein interactions is an important step in our quest to understand how the information contained in a genome is put into action. Although a number of experimental techniques can report on the existence of a protein–

to spot pattern databases for the comparison of electrophoresis results and genome

A large and growing application of 2-DE in "proteome analysis." Proteome analysis is "the analysis of the entire Protein complement expressed by a genome". The analysis involves the systematic separation, identification, and quantification of many proteins simultaneously from a single sample. Two-dimensional electrophoresis is used e due to its unparalleled ability to separate thousands of proteins simultaneously. Two-dimensional electrophoresis is also unique in its ability to detect post- and co-translational modifications, which cannot be predicted from the genome sequence. Applications of 2-DEinclude proteome analysis, cell differentiation, and detection of disease markers, monitoring therapies, drug discovery,

"Proteomics" is the large-scale screening of the proteins of a cell, organism or biological fluid, a process, which requires stringently controlled steps of sample preparation, 2-DE, image detection and analysis, spot identification, and database searches. Moreover, Proteomics studies lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, developmental stages etc. Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression. A keystone of conventional proteomics is high-resolution 2D gel electrophoresis followed by protein

As a technique with high-flux and high resolution, proteomics has been widely applied in proteome analysis of tumors. The onset and development of the tissues and cells can be detected at the entire protein level through analyzing the differential expression of proteins. The combination of 2-DE and mass spectrometry can be used to identify differential proteins between tumor cells and normal original cells, and these differential proteins imply a large quantity of biological information. Some of the special proteins are special markers of tumors. The most consistently successful proteomic method is the combination of twodimensional gel electrophoresis (2DE) for protein separation, visualization, and mass spectrometric (MS) identification of proteins using peptide mass fingerprints and tandem

The experiments form the basis of proteomics, and present significant challenges in data analysis, storage and querying. The core technology of proteomics is 2-DE. At present, there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure. The replacement of classical first-dimension carrier ampholyte pH gradients with well-defined immobilized pH gradients has resulted in higher resolution, improved inter-laboratory reproducibility, higher protein loading capacity, and an extended basic pH limit for 2-DE. With the increased protein capacity, micropreparative 2-DE has accelerated spot identification by mass spectrometry and Edman sequencing. The remarkable improvements in 2-DE resulting from immobilized pH gradient gels, together with convenient new instruments for IPG-IEF, will make critical contributions to advances

A comprehensive understanding of protein–protein interactions is an important step in our quest to understand how the information contained in a genome is put into action. Although a number of experimental techniques can report on the existence of a protein–

sequence databases for assignment of sequence information.

cancer research, purity checks, and microscale protein purification.

identification using mass spectrometry.

MS peptide sequencing.

in proteome analysis.

protein interaction, very few can provide detailed structural information. NMR spectroscopy is one of these, and in recent years several complementary NMR approaches, including residual dipolar couplings and the use of paramagnetic effects, have been developed that can provide insight into the structure of protein–protein complexes.

Two-dimensional gel electrophoresis for separation of complex protein samples coupled with mass spectrometry for protein identification has been used to analyze protein expression patterns for many sample types. Inherent in the use of this technique is information on not only full-length protein expression, but expression of modified, splice variant, cleavage product, and processed proteins. Any protein modification that leads to a change in overall protein charge and/or molecular weight (MW) will generate a different spot on the 2-DE. Modification specific staining can identify whether a specific posttranslational modification is responsible for the shift, and mass spectrometry can potentially identify the source of isoelectric point (pI) and/or MW differences. Due to the lack of complete coverage for a protein's amino acid sequence using either matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or high-performance liquid chromatography (HPLC) tandem mass spectrometry (LC-MS/MS), there has been limited success in using MS to identify isoforms and post-translational modifications. While the theoretical MW is often slightly higher than the MW of the fully processed protein due to cleavage of signal and pro-peptides, there can also be post-translational modifications that increase the protein's gel MW. Thus, an exploration into the causes of the difference in the theoretical MW and the MW as seen in the gel can yield information about the state of the protein. When the gel MW of a given protein is significantly lower than the calculated weight, the gel spot represents a protein fragment. The extent to which proteins are present as fragments or variants in tissues and fluids has not been determined, but the combination of 2-DE, Western blotting, and mass spectrometry–based protein identification makes such analyses possible. Two-dimensional gel electrophoresis of human mammary tissue, followed by immune blotting, resulted in multiple spots at significantly differing molecular weights. The function of protein fragments is dependent on activation processes and localization properties. This Chapter will be critically analyzed as per the contents given in the synopsis with up-to-date informations.
