Capillary electrophoresis analytical protein application

P. D. Grossman, J. C. Colburn, H. H. Lauer, R. G. Nielsen, R. M. Riggin, G. S. Sittampalam and E. C. Rickard, Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides . Anal. Chem. 61 1186-1194 (1989). 

Capillary electrophoresis systems are also likely to play an increasingly prominent analytical role in the QC laboratory (Figure 7.2). As with other forms of electrophoresis, separation is based upon different rates of protein migration upon application of an electric field. 

The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objective but capillary gel electrophoresis, including iso-electric focusing and SDS electrophoresis, is particularly useful for protein applications. 

Electrospray (ESI) is an atmospheric pressure ionization source in which the sample is ionized at an ambient pressure and then transferred into the MS. It was first developed by John Fenn in the late 1980s [1] and rapidly became one of the most widely used ionization techniques in mass spectrometry due to its high sensitivity and versatility. It is a soft ionization technique for analytes present in solution therefore, it can easily be coupled with separation methods such as LC and capillary electrophoresis (CE). The development of ESI has a wide field of applications, from small polar molecules to high molecular weight compounds such as protein and nucleotides. In 2002, the Nobel Prize was awarded to John Fenn following his studies on electrospray, for the development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules. ... 

One of the most powerful separation techniques for purity and impurity analysis for the bioanalyst is capillary electrophoresis (CE). However, it is a relatively new analytical tool and its methodology is evolving at a rapid pace, so there is limited reference to its application to protein impurity analysis in the literature. Nonetheless, this is only a temporary respite. In the future, CE will become a standard and routine analytical technique for the analysis of protein impurities in recombinant pharmaceuticals. 

Electrospray mass spectrometry has developed into a well-established method of wide scope and potential over the past 15 years. The softness of electrospray ionization has made this technique an indispensable tool for biochemical and biomedical research. Electrospray ionization has revolutionized the analysis of labile biopolymers, with applications ranging from the analysis of DNA, RNA, oligonucleotides, proteins as well as glycoproteins to carbohydrates, lipids, gly-colipids, and lipopolysaccharides, often in combination with state-of-the-art separation techniques like liquid chromatography or capillary electrophoresis [1,2]. Beyond mere analytical applications, electrospray ionization mass spectrometry (ESMS) has proven to be a powerful tool for collision-induced dissociation (CID) and multiple-stage mass spectrometric (MSn) analysis, and - beyond the elucidation of primary structures - even for the study of noncovalent macromolecular complexes [3]. 

During the last decade, capillary electrophoresis (CE) has developed into a widely applied method for the analysis of pharmaceuticals (both for the evaluation of pharmaceutical formulations and metabolites). These applications established the basis for introducing CE into the forensic held also. Today, capillary electrophoresis can be applied to a number of analytical problems in forensic science, including the analysis of gunshot residues, explosives, inks, dusts, soils, and, of course, illicit drugs, diverse toxicants, DNA hngerprinting, protein analysis, and so forth (for reviews, see Refs. 1 and 2). 

The application area of LC-MS is rapidly growing. LC-MS is now regularly used for the analysis of many different types of compound drugs and metabolites, herbicides-pesticides and metabolites, surfactants, dyes, saccharides, lipids-phospholipids, steroids, and many others. In our opinion, the area that profits more from the development of LC-MS is bioanalysis natural products, proteins, peptides, nucleosides, and metabolic studies. Despite the current trends toward immunoassays-biospecific assays and capillary electrophoresis, LC-MS is an extremely powerful analytical technique that is considered complementary to the above mentioned, rather than competitive. 

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

Immunoaffinity capillary electrophoresis is rapidly growing in the field of pharmaceutical and diagnostic applications. Several applications have been developed to affinity concentrate proteins and peptides found at low concentrations in simple and complex matrices in order to enhance analyte detectability when separated by CE (68,120,407,435). Figure 12 shows a microphotograph... 

Capillary electrophoresis has a wide applicability. High molecular weight compounds such as proteins, nucleic acids and oligosaccharides can be separated as well as smaller biomolecules such as peptides and amino acids. CE is not restricted to charged analytes. Neutral molecules can be separated from each other by employing a variation of CE called micellar electrokinetic chromatography (MEKC). This is frequently used for the separation of chiral drugs in pharmaceutical research. 

Grossman, P.D. et al. Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides, AnaZ. Chem., 61, 1186, 1989. 

Figure 7 CZE separation of hGH and its derivatives. CZE performed in FS capiiiary (i.d. 50 pm, totai iength 105cm, effective iength (to detector) 81.5cm BGE lOmmoii" Tricine, 5.8mmoii morphoiine, 20mmoii NaCi, pH8.0 eiectric fieid intensity 300Vcm current 20pA, temperature 24°C. 1, hGH 2, (desamido-149)- and (desamido-152)-hGH 3, (didesamido-149-152)-hGH. Mesityl oxide, electroosmotic fiow marker. (Reprinted with permission from Grossman PD, Coiburn JC, Lauer HH, et al. (1989) Application of free-solution capillary electrophoresis to the anaiyticai scaie separation of proteins and peptides. Analytical Chemistry 61(11) 1186-1194 American Chemical Society.)...

Microchips offer various possibilities for the performance of analytical assays they comprise a capillary system, to which electric fields can be applied, which is the principle of capillary electrophoresis (CE). Thus, capillary electrophoretic separation assays can be transferred to microchips leading to the accelerated separation of compounds, in particular of carbohydrates, amino acids, peptides, proteins, and nucleic acids. In Table 3, some representative assays are summarized with special emphasis on the practical applications. 

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