Title: Proteomic Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis
Abstract: The chapter focuses on proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and its applications. The proteomes from distinct samples are extracted and separated by 2D-PAGE and the relative abundance of each protein in the different samples is measured by visualizing the protein spots. After excising spots with different abundances between the two gels, the spots are enzymatically digested, and the extracted peptides are analyzed by mass spectrometry (MS) or tandem MS. These data are used in conjunction with an appropriate database to identify the protein. Once proteins are separated via 2D-PAGE, they need to be visualized before MS analysis. Proteins in electrophoretic gels can be visualized by a number of staining techniques. Visualizing the protein spots provides two key parameters: the ability to locate the protein within the gel and to compare its intensity with its spot on another gel so that a sense of its relative expression between two samples can be ascertained. In addition to the conventional staining techniques described in the chapter, methods have been developed for the specific labeling or staining of proteins that are glycosylated, phosphorylated, S-nitrosylated, adenosine diphosphate (ADP)-ribosylated, arginine methylated, and proteolytically modified. The key characteristics required of any effective staining technique are sensitivity, high linear-dynamic range for quantitation measurements, and compatibility with downstream MS analysis. Although the basic foundation of 2D-PAGE in proteomics is the high-resolution separation and quantitation of proteins from different samples, the explosion of proteomics has resulted in many different variations on this approach. Several applications of 2D-PAGE—such as effects of hypoxia on kidney protein expression, characterization of human prion protein by 2D-PAGE, proteome analysis of esophageal cancer by differential in-gel electrophoresis (DIGE), and proteome analysis of secreted proteins—are also presented
Publication Year: 2003
Publication Date: 2003-01-01
Language: en
Type: review
Indexed In: ['crossref', 'pubmed']
Access and Citation
Cited By Count: 10
AI Researcher Chatbot
Get quick answers to your questions about the article from our AI researcher chatbot