Fully automated two-dimensional capillary electrophoresis for high sensitivity protein analysis.
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.
Michels
David A
DA
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
Hu
Shen
S
Schoenherr
Regine M
RM
Eggertson
Michael J
MJ
Dovichi
Norman J
NJ
D000230:Adenocarcinoma / Q000737:chemistry; D001331:Automation; D019075:Electrophoresis, Capillary / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D019073:HT29 Cells; D006801:Humans; D007834:Lasers; D009363:Neoplasm Proteins / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009363:Neoplasm Proteins