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Trends in Chromatography   Volumes    Volume 5 
Application of two dimensional liquid chromatography to proteomics
Frédéric Pont
Pages: 1 - 14
Number of pages: 14
Trends in Chromatography
Volume 5 

Copyright © 2009 Research Trends. All rights reserved

To date, protein fractionation still represents a limiting step in a proteomic study and no single fractionation strategy has demonstrated the capacity to cover the whole proteome. Liquid chromatography has been used for years as a means of separation and it has become a powerful analytical technique. Chromatographic columns are in constant development and now it is possible to separate proteins with a high resolution using columns packed with small particle size silica or monolithic columns. Columns of different properties can be combined together to build a powerful multidimensional separation system. A good compromise to optimize both the resolving power and sample handling is bi-dimensional liquid chromatography. Indeed, two dimensional liquid chromatography systems have been developed for proteomic analysis and one system is now commercially available. This instrument separates proteins in the first dimension according to their pI using chromatofocusing, followed by a fractionation according to hydrophobicity, using reversed phase chromatography in the second dimension. An important panel of samples has been successfully analyzed using this technology.  An interesting feature of 2D-LC is that the proteins can be collected in liquid phase after fractionation. Interfacing 2D-LC with mass spectrometry is thus facilitated and many developments have been made in this field. Proteins have been analyzed with mass spectrometers on line, off line, before and after enzymatic cleavage. Quantitative approaches based on mass spectrometry have been combined with 2D-LC. An emerging application of 2D-LC is the building of protein microarrays. This review emphasizes two-dimensional liquid chromatography as a technology to study the proteome, discusses the capabilities and limitations of the current commercial instrument, and highlights the potential of two-dimensional liquid chromatography in proteomic analysis.
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