Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser‐induced fluorescence detection

Abstract

Previously, we have demonstrated postcolumn derivatization of proteins separated by capillary sieving electrophoresis (CSE), in which naphthalene-2,3-dicarbaldehyde was employed as a fluorogenic labeling reagent. Standard proteins separated by CSE were reacted with naphthalene-2,3-dicarbaldehyde in the presence of 2-mercaptoethanol (2-ME) which plays a role of a reducing agent in the derivatization reaction. To improve the sensitivity, we attempted the use of ethanethiol instead of 2-ME. Ethanethiol showed 1.4- to 4.5-fold lower limits of detection for proteins than 2-ME. Furthermore, we found that 8-aminopyrene-1,3,6-trisulfonate (APTS) is a good marker for relative electrophoretic mobilities of proteins in CSE. Since APTS is a fluorescent and trivalent anion, it generates strong fluorescence and migrates faster than any of the proteins. Therefore, we employed APTS as a marker to obtain the relative electrophoretic mobilities of proteins. The present method was applied to the analyses of proteins in biological samples. Human Ewing's family tumor cell line 'RDES' was used as a sample. The cultured cells were lysed with a buffer containing Tris-HCl, NaCl, sodium dodecyl sulfate, and 2-ME. After denaturation, the lysate was directly introduced into the capillary. Several peaks, which would correspond to proteins with molecular mass ranging from 10 to 93 kDa, were found in the cell lysate. In addition, we measured a milk sample by the CSE with postcolumn derivatization. The electropherogram showed five major peaks which corresponded to α-lactalbumin, β-lactoglobulin, κ-casein, bovine serum albumin, and mixture of α- and β-casein.

Protein separation by capillary gel electrophoresis: A review

“…The method used a labeling dye of naphthalene-2, 3-dicarbaldehyde in the presence of 2-mercaptoethanol which played a role of a reducing agent in the derivatization reaction. Recently, these researchers replaced 2-mercaptoethanol with ethanethiol as the reducing agent and improved the method limits of detection by 1.4- to 4.5-fold [109]. …”

“…As the SDS-CGE method was able to achieve the same conclusion as with SDS-PAGE, except for the patient with elliptocytosis, Debaugnies et al concluded that the new SDS-CGE method could be proposed as an automated alternative method to the labor-intensive SDS-PAGE analysis. Kaneta et al [109] applied CGE with postcolumn derivatization/LIF detection to analyses of two biological samples, namely a cell lysate and a milk sample.…”

Laser‐Induced Fluorometry for Capillary Electrophoresis

“…When using BGE containing 100 mM N ‐cyclohexyl‐2‐aminoethanesulfonic acid, DEAE, 0.1 % sodium dodecyl sulfate (SDS), and 1.5 % PEO (pH 8.6), six standard proteins treated by SDS could be separated according to their molecular mass. The BGE permitted the separations of standard proteins, proteins in a milk sample, and proteins in cancer cells by CSE, followed by post‐column derivatization with NDA …”

Discrimination of glycoproteins via two‐color laser‐induced fluorescence detection coupled with postcolumn derivatization in capillary electrophoresis

“…To solve that problem, postcolumn and oncolumn derivatizations are useful since native proteins can be injected directly into a capillary with no labeling. We also reported the postcolumn derivatization of proteins separated by capillary sieving electrophoresis, which permitted direct injection of denatured proteins in LIF detection .…”