Capillary electrophoresis library analysis

Boutin, J. A., Hennig, P., Lambert, P. H., et al. (1996) Combinatorial peptide libraries Robotic synthesis and analysis by nuclear magnetic resonance, mass spectrometry, tandem mass spectrometry, and high-performance capillary electrophoresis techniques. Anal. Biochem. 234, 126-141. 

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. 

Capillary electrophoresis (CE) is a powerful separation technique. It is especially useful for separation of ionic compounds and chiral mixtures. Mass spectrometry has been coupled with CE to provide a powerful platform for separation and detection of complex mixtures such as combinatorial libraries. However, the full potential of CE in the application of routine analysis of samples has yet to be realized. This is in part due to perceived difficulty in the use of the CE technique compared to the more mature techniques of HPLC and even SFC. Dunayevskiy et al. [136] analyzed a library of 171 theoretically disubstituted xanthene derivatives with a CE/ESI-MS system. The method allowed the purity and makeup of the library to be determined 160 of the expected compounds were found to be present, and 12 side products were also detected in the mixture. Due to the ability of CE to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple CE-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components. Boutin et al. [137] used CE-MS along with NMR and MS/MS to characterize combinatorial peptide libraries that contain 3 variable positions. The CE-MS method was used to provide a rapid and routine method for initial assessment of the construction of the library. Simms et al. [138] developed a micellar electrokinetic chromatography method for the analysis of combinatorial libraries with an open-tube capillary and UV detection. The quick analysis time of the method made it suitable for the analysis of combinatorial library samples. CE-MS was also used in the analysis... 

B. Volland, J.P f auchbre, J.L. Combinatorial Peptide Libraries Robotic Synthesis and Analysis by Nuclear Magnetic Resonance, Mass Spectrometry, Tandem Mass Spectrometry, and High-performance Capillary Electrophoresis Techniques, AnflZ. Biochem. 234, 126-141 (1996). 

For library analysis of compound mixtures, reliable results are obtained by coupling mass spectrometry with HPLC or capillary electrophoresis (CE). In this arrangement, mixtures of several hundred compounds can be analyzed. A comparison of MS, CE, and NMR for library characterization has been made in [93]. It was concluded that existing analytical techniques can provide sufficient information about prepared libraries. 

An additional dimension to library analysis is introduced when mass spectrometry is coupled with common separation techniques, for example liquid chromatography (LC) and capillary electrophoresis (CE). While these couplings are compatible with spray ionization techniques such as ESI or APCI, they more or less exclude the use of the MALDI technique. Several contributions deal with LC-ES-MS [36, 37] and CE-ES-MS-coupling [38], For molecules with isobaric nominal masses, MS/MS-experiments are performed to confirm the identity of a library component. Separation and analysis of compound mixtures may also be performed by GC-MS [39, 40], As a supplement to the more common-... 

It maybe expected that the different kind of MIP already in use in various areas of chemical analysis will find their application as well on the field of capillary electrophoresis, e.g., MIP in CEC for screening combinatorial libraries, as firstly published by Vallano and Remcho [16] who used packed capillaries for screening tricyclic antidepressants. 

Figure 9.8 NECEEM-based selection of smart aptamers with predefined A off. (A) Two aptamer-collection windows with respect to the migration time of the naive DNA library. The intact complexes (not shown) elute in region I. Region II corresponds to aptamers that dissociated from the complexes during electrophoresis. (B) NECEEM-based affinity analysis of enriched libraries after two rounds of selection in regions I (left) and II (right). Experiments depicted in part (B) were performed with a shorter capillary than experiments shown in part (A) accordingly, migrations times are shorter in (B).

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