Proteomics isoelectric focusing

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Jensen, P. K., Pasa-Tolic, L., Anderson, G. A., Homer, J. A., Lipton, M. S., Bmce, J. E., and Smith, R. D. (1999). Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 71, 2076-2084. 

Breci, L., Hattrup, E., Keeler, M., Letarte, J., Johnson, R., Haynes, PA. (2005). Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing. Proteomics 5, 2018-2028. 

Chen, J., Balgley, B.M., DeVoe, D.L., Lee, C.S. (2003a). Capillary isoelectric focusing-hased multidimensional concentration/separation platform for proteome analysis. Anal. Chem. 

Chen, J., Gao, J., Lee, C.S. (2003b). Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies. J. Proteome Res. 2, 249-254. 

Chen, J., Lee, C.S., Shen, Y., Smith, R.D., Baehrecke, E.H. (2002). Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23, 3143-3148. 

Essader, A.S., Cargile, B.J., Bundy, J.L., Stephenson, J.L., Jr. (2005). A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics. Proteomics 5, 24—34. 

Wang W, Guo T, Rudnick PA, et al. Membrane proteome analysis of micro-dissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS. Anal. Chem. 2007 79 1002-1009. 

Balgley BM, Wang W, Song T, et al. Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing-based proteomic platform coupled with a spectral counting approach. Electrophoresis 2008 29 3047-3054. 

L. Breci, E. Hattrup, M. Keeler, J. Letarte, R. Johnson, and P. A. Haynes. Comprehensive Proteomics in Yeast Using Chromatographic Fractionation, Gas Phase Fractionation, Protein Gel Electrophoresis, and Isoelectric Focusing. Proteomics, 5(2005) 2018-2028. 

Coverage The use of large format gels, pre-firactionation and multiple use of narrow range isoelectric focusing gels enable more comprehensive proteomic studies... 

Gorg A, Boguth G, Kopf A, ReU G, Parlar H, Weiss W. (2002) Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels. Proteomics 2, 1652-7. 

Shang TQ, Ginter JM, Johnston MV, Larsen BS, McEwen CN. Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures. Electrophoresis 2003 24 2359-2368. 

Kennedy S (2002) The role of proteomics in toxicology identification of biomarkers of toxicity by protein expression analysis. Biomarkers 7 269-290 Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Hu-mangenetik 26 231-243... 

In spite of all these clear advantages, capillary isoelectric focusing is still not used as a routine method in bioanalysis, although more and more papers show important applications. Study of the proteome combined with microchip technology may, however, cause sudden progress in this innovative technique. In the past decade several reviews have appeared about CIEF separations [5-17]. This chapter summarizes the theory and the newest innovations, as well as applications of CIEF. 

Capillary isoelectric focusing will be applied mostly for proteome studies in the future. Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis is by far too slow for a... 

Capillary isoelectric focusing has proven its separation potential and special advantages in proteome bioanalysis. Several applications have been reported using this unique methodology. Notwithstanding, further improvements are necessary to design fully automated chip-based devices and a more advanced coupling to various detection systems. 

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