Capillary isoelectric focusing protein analysis

Manning JM et aJ Normal and abnormal protein subunit interactions in hemoglobins.] Biol Chem 1998 273 19359-Mario N, Baudin B, Giboudeau J Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing. J Chromatogr B Biomed Sci Appl 1998 706 123-Reed W, Vichinsky EP New considerations in the treatment of sickle cell disease. Annu Rev Med 1998 49 46l. 

Tang, Q., Harrata, A. K., Lee, C. S. (1997). Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69(16), 3177-3182. 

High-efficiency separations of FQ-labeled proteins are only achieved in the presence of an anionic surfactant, such as SDS. As a result, capillary isoelectric focusing is not useful for the analysis of these proteins. Instead, we employ capillary sieving electrophoresis and micellar electrokinetic capillary chromatography for our two-dimensional electrophoresis. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

The on-line interfacing of capillary isoelectric focusing with Fourier-transform ion cyclotron resonance-mass spectrometry (FTICR-MS) was shown to be effective for separating minor components of protein mixtures for on-line mass spectral analysis [62-64],... 

Capillary isoelectric focusing can be applied as a micropreparative tool for protein analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) [69,70]. The exact timing of the collector steps in the interface is based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. During the collection a sheath flow fraction collector is used to maintain the permanent electric current. 

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

Q. Tang, A.K. Harrata and C.S. Lee, Capillary isoelectric-focusing electrospray mass-spectrometry for protein analysis. Analytical Chemistry, 67, 3515-3519 (1995). 

N.J. Clarke and S. Naylor, Capillary isoelectric focusing-mass spectrometry analysis of protein mixtures from human body fluids, Biomedical Chromatography, 16, 287-297 (2002). 

Schwer C (1995). Capillary isoelectric focusing a routine method for protein analysis Electrophoresis. 16 2121-2126. 

Kuroda Y, YukinagaH, Kitano M, Noguchi T, NematiM, ShibukawaA, NakagawaT, Matsuzaki K. Online capillary isoelectric focusing-mass spectrometry for quantitative analysis of peptides and proteins. J Pharm Biomed Anal 2005 37 423-8. 

Wei, J., L. Yang, A. K. Harrata, and C. S. Lee, High resolution analysis of protein phosphorylation using capillary isoelectric focusing—electrospray ionization—mass spectrometry. Electrophoresis, 19, 2356-2360, 1998. 

Liu, Z. and Pawliszyn, J., Coupling of solid-phase microextraction and capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection for protein analysis. Analytical Chemistry, 77,165, 2005. 

Table 3.2 summarizes the most popular proteome profiling techniques. Recent developments introduced capillary electrophoresis [92] not only for DNA analysis and sequencing, but also for high-resolution protein separation, based on their size (capillary electrophoresis-sodium dodecyl sulfate [CE-SDS]) and capillary isoelectric focusing (cIEF) point... 

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