Capillary electrophoresis amino acid analysis

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Smith, J. T., Recent advances in amino acid analysis using capillary electrophoresis, Electrophoresis, 20, 3078, 1999. 

Cheng, Y.-F. and Dovichi, N. J., Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence, Science, 242, 562, 1988. 

Zahou, E., Jornvall H., and Bergman T., Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation, Anal. Biochem., 281, 115, 2000. 

K. Arlt, S. Brandt, and J. Kehr, Amino acid analysis in five pooled single plant cell samples using capillary electrophoresis coupled to laser induced fluorescence detection. J. Chromatogr. A 926, 319 325 (2001). 

Acid hydrolysis under standard conditions (6M HC1, 110 °C, 24 h) leads to partial decomposition of selenocystine and selenocysteine derivatives, thus making quantification of this amino acid by amino acid analysis difficult. Similarly, acid hydrolysis of 5e-[2-(4-pyr-idinyl)ethyl]selenocysteine peptides, obtained by reduction of the selenocystine peptides with NaBH4 and reaction with 4-vinylpyridine, results in partial decomposition. This de-rivatization, however, is useful for the enantiomeric resolution of the acid hydrolysates by capillary zone electrophoresis by applying host-guest complexation with crown ethers.11" 22 ... 

Because of these ever-widening interests, the measurement of plasma tHcy is undertaken in many clinical chemistry and routine laboratories. Various methods are employed, including high-performance liquid chromatography (HPLC) assays, conventional amino acid analysis, capillary electrophoresis, gas chromatography with or without mass spectrometry, liquid chromatography with tandem mass spectrometry, and in many routine clinical chemistry laboratories immunoassays. In this chapter, those methods that are often available in laboratories involved in the investigation of inborn errors of metabolism are described, namely HPLC and tandem mass spectrometry. 

As for all synthetic products to be tested in biological systems, a careful analytical characterization of peptide libraries is crucial in order to confirm their identity and establish their quality. Compared to individual peptides, however, the analysis of peptide libraries is complicated due to the fact that the peptides are either bound to a solid support or arranged in highly complex mixtures. This poses certain restrictions on which analytical methods can be used to characterize combinatorial libraries. For example, analytical methods that are based on the separation of product components, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), are only of limited use for the analysis of peptide libraries, in particular of those made up of complex nnixtures (>100 peptides per mixture). The analytical methods beneficially applicable to peptide libraries include amino acid analysis, mass spectrometry, and sequencing. 

Figure 12.4. Laser-induced fluorescence detector for CZE.4 [Reprinted, with permission, from Y.-F. Cheng and N. J. Dovichi, Science 242, 1988, 562-564. Subattomole Amino Acid Analysis by Capillary Zone Electrophoresis and Laser-Induced Fluorescence . Copyright 1988 by A A AS.

Modern tHcy methods include enzyme immunoassays and chromatographic-based methods. In practice, immunoassays " are most often used for routine purposes (e.g., fluorescence polarization immunoassay IFPIA] as run on Abbott s IMx and AxSYM platforms) Chromatographic assays include amino acid analysis high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection ° " capillary electrophoresis with fluorescence detection gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem MS (MS-MS). 

Besides amino acid analysis and elaborated mass spectroscopy techniques, many more analytical methods are applied to support the identity examinations of the protein drug, such as determination of the extinction coefficient, isoelectric point, and crystal structure, as well as recording the nuclear magnetic resonance (NMR) and circular dichroism (CD) spectra and determining the chromatographic profiles from HPLC-runs as well as from capillary and polyacrylamide gel electrophoresis (CE and PAGE, respectively). 

Poinsot, V., Lacroix, M., Maury, D., Chataigne, G, Feurer, B., and Couderc, F. Recent advances in amino acid analysis by capillary electrophoresis. Electmphoresis, 27,176, 2006. 

Soga, T., and Heiger, D.N., Amino acid analysis hy capillary electrophoresis electrospray ionization mass spectrometry. Ana/. Chem., 72, 1236, 2000. 

Figure 2 CE-ESI-MS selected ion electropherograms for amino acids in soy sauce. Experimental conditions fused-silica capillary 50 urn i.d. X 100 cm electrolyte, 1 mol r formic acid applied voltage, 30kV injection 3 sat 50 mbar temperature, 20°C sheath liquid, lOnlmin" of 5moll ammonium acetate in 50% (v ) methanol-water. (Reprinted with permission from SogaT and Heiger DN (2000) Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Analytical Chemistry 72 1236-1241 American Chemical Society.)...

My research group has focused on the use of very high sensitivity fluorescence detection in capillary electrophoresis for the analysis of amino acids [35-47], for DNA sequencing [37, 38], for analysis of proteins [39, 40], for analysis of enzymes [41, 42], and for analysis of sugars [43, 44], These systems provide detection limits from a few hundred molecules down to single molecules of fluorescent analyte. Quite complex mixtures can be separated and analyzed the present state-of-the-art appears to be resolution of over 600 DNA sequencing fragments in a two hour separation [38]. 

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