Proteomics Peptides

Popa, T.V., Mant, C.T., and Hodges, R.S. Ion interaction capillary zone electrophoresis of cationic proteomic peptide standards. 7. Chromatogr. A. 2006, 1111 192-199. 

Since selectivity is a prerequisite for resolution, a temperature increase strongly impacts both selectivity and resolution. The variability of the van t Hoff lines explains the seemingly erratic result of the influence of tanperature on the figures of merit of the method. For example, at lower temperatures, the resolution of model peptides with +1 and +3 net charges improved and worsened, respectively [27]. In chiral IPC, T < 0°C was successfully used to improve enantioresolution [28]. Similarly, lower temperatures provided better resolution in the analysis of a new aminoglycoside antibiotic [29] and for characterization of maize products [30]. Conversely, an increased resolution at 70°C was observed when the ion-pair mechanism was exploited under IPC-capillary zone electrophoresis of cationic proteomic peptide standards [31]. 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Kaliszan, R., Baczek, T., Cimochowska, A., Juszczyk, P., Wisniewska, K., Grzonka, Z. Prediction of high-performance liquid chromatography retention of peptides with the use of quantitative structure-retention relatiorrships. Proteomics 2005, 5, 409 15. 

The use of CIEF in combination with FTICR has been demonstrated in an analysis of the E. coli proteome (Jensen et al., 1999). For these experiments, E. coli was grown in a medium depleted of rare isotopes in order to increase the mass measurement accuracy. The high abundance isotopes are present at approximately 98.89% 12C, 99.63% 14N and 99.985% H. For peptides, the presence of rare isotopes does not significantly change the spectra but with undigested proteins, mass accuracy can be limited by the broadened distribution of ions of any given protein due to the incorporation... 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Ji, J. Chakraborty A. Geng, M. Zhang, X. Amini, A. Bina, M. Regnier, F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B Biomed. Sci. Appl. 2000, 745,197-210. 

Proteomics algorithms have been developed to search databases of protein sequences for matches to short sequences determined experimentally from proteins or peptides in the laboratory,5 59 and for theoretical matches to... 

Although the feasibility of the proteomics or bioinformatics approach has been demonstrated, considerable room remains for improved methods for selective solublization of protein biomarkers and for rapid cleavage to produce peptides. There is also demand for advanced instrumentation to collect, process, and analyze microorganisms. 

FIGURE 9.1 Liquid chromatography workflow strategy options in proteomics. (a) bottom-up approach (b) top-down approach (c) selective sample cleanup directly combined with chromatographic separation (d) peptide capture with affinity restricted access material. 

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