Femtomolar concentration detection limit and zeptomole mass detection limit for protein separation by capillary isoelectric focusing and laser-induced fluorescence detection

Abstract

Fluorescence tends to produce the lowest detection limits for most forms of capillary electrophoresis. Two issues have discouraged its use in capillary isoelectric focusing. The first issue is fluorescent labeling of proteins. Most labeling reagents react with lysine residues and convert the cationic residue to a neutral or anionic product. At best, these reagents perturb the isoelectric point of the protein. At worse, they convert each protein into hundreds of different fluorescent products that confound analysis. The second issue is the large background signal generated by impurities within commercial ampholytes. This background signal is particularly strong when excited in the blue portion of the spectrum, which is required by many common fluorescent labeling reagents. This paper addresses these issues. For labeling, we employ Chromeo P540, which is a fluorogenic reagent that converts cationic lysine residues to cationic fluorescent products. The reaction products are excited in the green, which reduces the background signal generated by impurities present within the ampholytes. To further reduce the background signal, we photobleach ampholytes with high-power photodiodes. Photobleaching reduced the noise in the ampholyte blank by an order of magnitude. Isoelectric focusing performed with photobleached pH 3-10 ampholytes produced concentration detection limits of 270 +/- 25 fM and mass detection limits of 150 +/- 15 zmol for Chromeo P540 labeled beta-lactoglobulin. Concentration detection limits were 520 +/- 40 fM and mass detection limits were 310 +/- 30 zmol with pH 4-8 ampholytes. A homogenate was prepared from a Barrett's esophagus cell line and separated by capillary isoelectric focusing, reproducibly generating dozens of peaks. The sample taken for the separation was equal to the labeled protein homogenate from three cells.

Figure 1

Isoelectric focusing profile of pH 4–8 ampholyte blanks. The top curve is the untreated blank, the middle curve is after 2 minutes of photobleaching, and the bottom trace is after 30 minutes of photobleaching. Isoelectric focusing was performed in a Gaurant-coated capillary. Data not offset.

Figure 2

Capillary isoelectric focusing analysis of Chromeo p540-labeled β-lactoglobulin in photobleached pH 4–8 ampholytes. Isoelectric focusing was performed in a Gaurant coated capillary. Data offset for clarity.

Figure 3

Capillary isoelectric focusing analysis of Chromeo p540-labeled β-lactoglobulin in photobleached pH 3–10 ampholytes. Isoelectric focusing was performed in an acrylamide-coated capillary. Data offset for clarity.

Figure 4

Capillary isoelectric focusing analysis of protein homogenate in pH 3–10 ampholytes. The amount of labeled protein taken for analysis corresponds to the content of ~3 cells, except for the top trace, which had been diluted by a factor of two; the data were multiplied by a factor of two to correct for the dilution. Separations performed in a Gaurant-coated capillary. Data offset for clarity.

Figure 5

Capillary isoelectric focusing analysis of protein homogenate in pH 4–8 ampholytes. The amount of labeled protein taken for analysis corresponds to the content of ~3 cells. Separations were performed in a Gaurant coated capillary. Data offset for clarity.