Fully Automated Two-dimensional Capillary Electrophoresis for High Sensitivity Protein Analysis

• Published in: Molecular & cellular proteomics Vol. 1; no. 1; pp. 69 - 74
• Main Authors: Michels, David A, Hu, Shen, Schoenherr, Regine M, Eggertson, Michael J, Dovichi, Norman J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01.01.2002
• Subjects: Adenocarcinoma - chemistry; Automation; Electrophoresis, Capillary - methods; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; HT29 Cells; Humans; Lasers; Neoplasm Proteins - analysis; Neoplasm Proteins - isolation & purification; Sensitivity and Specificity; Spectrometry, Fluorescence
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.T100009-MCP200

We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity.