Peptides zone electrophoresis

Figure 9.10 Three-dimensional representation of the data volume of a tryptic digest of ovalbumin. Series of planar slices through the data volume produce stacks of disks in order to show peaks. Reprinted from Analytical Chemistry, 67, A. W. Moore Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatogra-phy/reversed phase liquid chromatography/optically gated capillary zone electrophoresis, pp. 3456-3463, copyright 1995, with permission from the American Chemical Society.

A. W. Moore, Jr and J. W. Jorgenson, Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/ optically gated capillary zone electrophoresis . Anal. Chem. 67 3456-3463 (1995). 

Nielsen, R. G., Riggin, R. M., and Rickard, E. C., Capillary zone electrophoresis of peptide fragments from trypsin digestion of biosynthetic human growth hormone, /. Chromatogr., 480, 393, 1990. 

Mosher, R. A., The use of metal ion-supplemented buffers to enhance the resolution of peptides in capillary zone electrophoresis, Electrophoresis, 11, 765, 1990. 

Grossman, R D., Wilson, K. J., Petrie, G., and Lauer, H. H., Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis, Anal. Biochem., 173, 265, 1988. 

Heegard, N. H. H. and Robey, R A., Use of capillary zone electrophoresis to evaluate the binding of anionic carbohydrates to synthetic peptides derived from human serum amyloid P component, Anal. Chem., 64, 2479, 1992. 

Righetti, P.G., Gelfi, C., Perego, M., Stoyanov, A.V., and Bossi, A., Capillary zone electrophoresis of oligonucleotides and peptides in isolectric buffers theory and methodology, Electrophoresis, 18, 2145, 1997. 

Liu H., Cho B.-Y., Strong R., Krull I.S., Cohen S., Chan K.C., and Issaq H.J., Derivatization of peptides and small proteins for improved identification and detection in capillary zone electrophoresis (CZE), Anal. Chim. Acta, 400, 181, 1999. 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

Table 1 summarizes several of the experimental methods discussed in this chapter. A need exists for new or revised methods for transport experimentation, particularly for therapeutic proteins or peptides in polymeric systems. An important criterion for the new or revised methods includes in situ sampling using micro techniques which simultaneously sample, separate, and analyze the sample. For example, capillary zone electrophoresis provides a micro technique with high separation resolution and the potential to measure the mobilities and diffusion coefficients of the diffusant in the presence of a polymer. Combining the separation and analytical components adds considerable power and versatility to the method. In addition, up-to-date separation instrumentation is computer-driven, so that methods development is optimized, data are acquired according to a predetermined program, and data analysis is facilitated. 

Moore, A.W., Jorgenson, J.W. (1995a). Rapid comprehensive two-dimensional separations of peptides via RP-HPLC-optically gated capillary zone electrophoresis. Anal. Chem. 67, 3448-3455. 

Sweedler reported a two-dimensional separation, where fluorescein thiocarbamyl derivatives of peptides were separated by capillary zone electrophoresis in the first dimension (Liu and Sweedler, 1996). The outlet of the capillary was wiped across the top of an SDS-PAGE gel, where peptides were then separated based on their size. 

These systems rely on various combinations of size-exclusion chromatography, reversed-phase chromatography, and zone electrophoresis to characterize amines, peptides, and proteins (Yamamoto etal., 1989 Bushey and Jorgenson 1990 Larmann et al., 1993, Moore and Jorgenson, 1995 Optick and Jorgenson, 1997). Haleem Issaq reviews these separations in Chapter 16 of this book. 

Zhao, J.Y., Waldron, K.C., Miller, J., Zhang, J.Z., Harke, H., Dovichi, N.J. (1992). Attachment of a single fluorescent label to peptides for determination by capillary zone electrophoresis. J. Chromatogr. 608, 239-242. 

Janini, G.M., Chan, K.C., Conrads, T.P., Issaq, H.J., Veenstra, T.D. (2004). Two-dimensional liquid chromatography-capillary zone electrophoresis—sheathless electrospray ionization-mass spectrometry evaluation for peptide analysis and protein identification. Electrophoresis 25, 1973-1980. 

Solinova V, Kasicka V, Koval D et al (2004) Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection. J Chromatogr B 808 75-82... 

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

Y. An, J. W. Cooper, B. M. Balgley, and C. S. Lee. Selective Enrichment and Ultrasensitive Identification of Trace Peptides in Proteome Analysis Using Transient Capillary Isotachophoresis/Zone Electrophoresis Coupled with Nano-ESI-MS. Electrophoresis, 27(2006) 3599-3608. 

C Miller, J Rivier. Peptide chemistry development of high performance liquid chromatography and capillary zone electrophoresis. Biopolymers (Pept Sci) 40, 265, 1996. 

D.J. Winzor, Classical approach to interpretation of the charge-dependence of peptide mobilities obtained by capillary zone electrophoresis../. ChromatogrA 1015 (2003) 199-204. 

Boss H J., Watson D.B., and Rush R.S. (1998), Peptide capillary zone electrophoresis mass spectrometry of recombinant human erythropoietin an evaluation of the analytical method, Electrophoresis 19(15), 2654—2664. 

Janini, G. M., Metral, C. J., Issaq, H. J., and Muschik, G. M. (1999). Peptide mobility and peptide mapping in capillary zone electrophoresis — experimental determination and theoretical simulation. J. Chromatogr. A 848, 417-433. 

Interactions between the solutes and the capillary wall also have a negative effect on the efficiency in capillary zone electrophoresis. Both hydrophobic interactions and electrostatic interactions of cations with the negatively charged capillary wall can be the cause of solute adsorption. Significant adsorption has been found for high-molecular-weight species, e.g., peptides and proteins. Because of the increased surface-area-to-volume ratio of narrow-bore capillaries, this effect is even more pronounced. 

MA Moseley, JW Jorgenson, J Shabanowitz, DF Hunt, KB Tomer. Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass ppectrometry and tandem mass spectrometry analysis of peptides. J Am Soc Mass Spectrom 3 289-300, 1992. 

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 ... 

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

Several different analytical and ultra-micropreparative CEC approaches have been described for such peptide separations. For example, open tubular (OT-CEC) methods have been used 290-294 with etched fused silicas to increase the surface area with diols or octadecyl chains then bonded to the surface.1 With such OT-CEC systems, the peptide-ligand interactions of, for example, angiotensin I-III increased with increasing hydrophobicity of the bonded phase on the capillary wall. Porous layer open tubular (PLOT) capillaries coated with anionic polymers 295 or poly(aspartic acid) 296 have also been employed 297 to separate basic peptides on the inner wall of fused silica capillaries of 20 pm i.d. When the same eluent conditions were employed, superior performance was observed for these PLOT capillaries compared to the corresponding capillary zone electrophoresis (HP-CZE) separation. Peptide mixtures can be analyzed 298-300 with OT-CEC systems based on octyl-bonded fused silica capillaries that have been coated with (3-aminopropyl)trimethoxysilane (APS), as well as with pressurized CEC (pCEC) packed with particles of similar surface chemistry, to decrease the electrostatic interactions between the solute and the surface, coupled to a mass spectrometer (MS). In the pressurized flow version of electrochromatography, a pLC pump is also employed (Figure 26) to facilitate liquid flow, reduce bubble formation, and to fine-tune the selectivity of the separation of the peptide mixture. 

Zone electrophoresis is mostly used for biological applications. Peptide separation and the measurement of protein fractions from blood serum (proteinogram of albumin and o-, (3- and 7-globulins) are among the better known applications. This TLC for biochemists is useful for the separation of polysaccharides, nucleic acids (for DNA sequencing), proteins and other colloidal species. 

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