Abstract
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.
Highlights
We report a system for automated protein analysis
If the two separation techniques are based on unrelated characteristics of the sample, the number of resolution elements is given by the product of the resolution elements of both separation steps
Column-switching technology is an alternative means of performing two-dimensional separations and is commonly used in chromatographic resolution of complex samples
Summary
Reagents—The labeling reagent FQ and cyanide are from Molecular Probes. 106 cells are lysed by sonication and labeled with 100 nmol of FQ in the presence of 2.5 mM potassium cyanide at 65 °C for 5 min. The FQ-labeled cell extract is diluted 50-fold in 10 mM phosphate buffer, pH 7, and stored on ice [15]. Separation is performed in two 40-cm-long, 50-m-inner diameter, 138-m-outer diameter polyamide-coated fused silica capillaries (Polymicro). Buffer 2 (CAPS) inlet and outlet capillaries have 145-m inner diameters/367-m outer diameters with lengths of 6 and 15 cm. The interface is rinsed with distilled water to remove any particles and air trapped in the cross. The capillaries are held in place by tightening the ferrules in the Valco cross
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