Application of Separation technologies to Proteomics Research

Abstract

Publisher Summary The chapter focuses on the application of separation technologies to proteomics research. Many instrumental strategies have been developed that are based on efficient separation followed by mass spectrometry (MS) identification and quantitation. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the method of choice for the separation of complex mixtures of proteins. In 2D-PAGE, proteins are separated in two sequential steps based on their charge and mass. 2D-PAGE has several limitations, including its ability to visualize only the highest abundant proteins within a proteomic mixture. Therefore, other sample preparation, concentration, separation, and detection methods need to be developed that would allow higher resolution than 2D-PAGE to separate a larger number of proteins, and the methods also need to be sensitive enough to detect the low-abundance proteins. Many separation methods have been reported in the scientific literature employing a single high-resolution procedure, as well as a multidimensional approach of high-pressure liquid chromatography (HPLC/HPLC) or HPLC/ capillary electrophoresis (CE) online or offline with MS for protein identification. The separation method selected depends primarily on the nature and complexity of the sample to be characterized. In addition, the focus of the proteome analysis also dictates the nature of the separation selected, as many separation techniques have been developed to extract specific classes of proteins. Regardless of the project focus, the continuing development of effective fractionation methods, both gel based and solution based, will be crucial to the continuing success of proteomics. The advantages and limitations of 2D-PAGE in comparison to other separation methods are also discussed.