UNIT 23.3 Proteomic Analysis Using 2-D Liquid Separations of Intact Proteins From Whole-Cell Lysates
Published Online: 1 FEB 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Zhu, K., Yan, F., O'Neil, K. A., Hamler, R., Lubman, D. M., Lin, L. and Barder, T. J. 2004. Proteomic Analysis Using 2-D Liquid Separations of Intact Proteins From Whole-Cell Lysates. Current Protocols in Protein Science. 34:23.3.1–23.3.28.
- Published Online: 1 FEB 2004
- Published Print: NOV 2003
This unit describes procedures for 2-D liquid separations of proteins from whole-cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI-based fractionation using a batch-phase electrophoretic method, while chromatofocusing uses a column-based chromatographic method to generate the pH gradient. Using either method, a second-dimension fractionation is provided in the liquid phase using nonporous silica-based reversed-phase HPLC (NPS-RP-HPLC) to generate a 2-D liquid map of the protein content of the cell. The eluate of the 2-D liquid fractionation is directly coupled to a mass spectrometer for on-line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.
- liquid phase IEF;
- protein separation;
- nonporous separations;