Two-dimensional capillary electrophoresis

TWO-DIMENSIONAL CAPILLARY ELECTROPHORESIS FOR THE COMPREHENSIVE ANALYSIS OF COMPLEX PROTEIN MIXTURES... 

Instrument We reported the first two-dimensional capillary electrophoresis system for protein analysis (Michels et al., 2002, Hu et al., 2004, Michels... 

FIGURE 15.4 Computer record of a two-dimensional capillary electrophoresis analysis of a protein homogenate prepared from ahiopsy obtained from the fundus of a Barrett s esophagus patient. The data were generated hy performing 1 s transfers between capillaries and a 9 s second-dimension separation. The first-dimension separation employed the same buffer as the CSE separation in Fig. 15.1 and the second-dimension separation employed the same buffer as the MECC separation in Fig. 15.1. 

We have analyzed the run-to-run reproducibility of the two-dimensional capillary electrophoresis system. The relative standard deviation in both MECC and CSE dimensions is better than 2% for the 50 most intense components in a set of five runs, and a simple gel alignment algorithm provides a twofold improvement in precision. 

Figure 15.10 presents the two-dimensional capillary electrophoresis fingerprint of a single MCF-7 breast cancer cell. This cellhad been fixed in 70% ethanol before analysis. 

FIGURE 15.10 Landscape image of a two-dimensional capillary electrophoresis analysis of the protein content of a single fixed MCF-7 breast-cancer cell. The data consist of a few high-amplitude components. 

Two-dimensional capillary electrophoresis of complex protein samples requires careful attention to detail. Tissues and cells should be fixed to prevent degradation. Most conventional fixatives are inappropriate because they produce covalent crosslinks, which are difficult to reverse. We find that ethanol produces decent results when the sample is homogenized with high concentrations of SDS. 

Chen, X., Wu, H., Mao, C., Whitesides, G.M. (2002). A prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane). Anal. Chem. 74, 1772-1778. 

Michels, D.A., Hu, S., Schoenherr, R.M., Eggertson, M.J., Dovichi, NJ. (2002). Fully automated two-dimensional capillary electrophoresis for high sensitivity protein analysis. Mol. Cell. Proteomics 1, 69-74. 

Becker, H., Lowack, K., Manz, A., Planar quartz chips with submicron channels for two-dimensional capillary electrophoresis applications.. /. Micromech. Microeng. 1998, 8, 24-28. 

Li, Y.H. Wojcik, R Dovichi, N.J. (2011). A replaceable microreactor for on-line protein digestion in a two dimensional capillary electrophoresis system with tandem mass spectrometry detection. Journal of Chromatograph / A, Vol.1218, No.l5 (April 2011), pp. 2007-2011, ISSN 0021-9673... 

Harwood MM, Christians ES, Fazal MA, Dovichi NJ. Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis. J Chromatogr A 2006 1130 190-4. 

Michels DA, Hu S, Dambrowitz KA, Eggertson MJ, Lauterbach K, Dovichi NJ. Capillary sieving electrophoresis-micellar electrokinetic chromatography fuUy automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate. Electrophoresis 2004 25 3098-105. 

Kraly JR, Jones MR, Gomez DG Dickerson JA, Harwood MM, Eggertson M, Paulson TG et al. Reproducible two-dimensional capillary electrophoresis analysis of Barrett s esophagus tissues. Anal Chem 2006 78 5977-86. 

Chemical Cytometry oe Proteins, Biogenic Amines, and Metabolic Cascades by One-and Two-Dimensional Capillary Electrophoresis... 

Gonzalez-Gomez, D., D. Cohen, J. A. Dickerson, X. Chen, F. Canada-Canada, and N. J. Dovichi. 2009. Improvement in protein separation in Barretts esophagus samples using two-dimensional capillary electrophoresis analysis in presence of cyclodextrins as buffer additives. Talanta 78 193-8. 

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