Reproducible Two-Dimensional Capillary Electrophoresis Analysis of Barrett's Esophagus Tissues

Abstract

We have constructed a high-speed, two-dimensional capillary electrophoresis system with a compact and high-sensitivity fluorescence detector. This instrument is used for the rapid and reproducible separations of Barrett's esophagus tissue homogenates. Proteins and biogenic amines are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled biomolecules are separated sequentially in two capillaries. The first capillary employs capillary sieving electrophoresis using a replaceable sieving matrix. Fractions are successively transferred to a second capillary where they undergo additional separation by micellar electrokinetic capillary chromatography. The comprehensive two-dimensional separation requires 60 min. Within-day migration time reproducibility is better than 1% in both dimensions for the 50 most intense features. Between-day migration time precision is 1.3% for CSE and better than 0.6% for MECC. Biopsies were obtained from the squamous epithelium in the proximal tubular esophagus, Barrett's epithelium from the distal esophagus, and fundus region of the stomach from each of three Barrett's esophagus patients with informed consent. We identified 18 features from the homogenate profiles as biogenic amines and amino acids. For each of the patients, Barrett's biopsies had more than 5 times the levels of phenylalanine and alanine as compared to squamous tissues. The patient with high-grade dysplasia shows the highest concentrations for 13 of the amino acids across all tissue types. Concentrations of glycine are 40 times higher in squamous biopsies compared to Barrett's and fundal biopsies from the patient with high-grade dysplasia. These results suggest that two-dimensional capillary electrophoresis may be of value for the rapid characterization of endoscopic and surgical biopsies.

Reaction of fluorogenic reagents with proteins

“…Capillary electrophoresis was performed on a locally constructed instrument employing postcolumn laser-induced fluorescence detection with a sheath-flow cuvette [10,[13][14]. Excitation was provided by a 473 nM laser (Lasermate Group, Pomona, CA, USA).…”

“…These products can have dramatically different electrophoretic behavior, leading to an envelope of peaks producing complex electropherograms [4][5][6]. Most of our work has been with FQ, which converts cationic lysine residues into neutral products, which can be detected with exquisite sensitivity [7][8][9][10]. More importantly, we have shown that the incorporation of an anionic surfactant, such as SDS, results in the collapse of the multiple labeling envelope into a single, sharp peak [7].…”

Two‐dimensional protein separation in microfluidic devices

“…High-resolution 2-D separation was achieved in 1990 by coupling HPLC with CE [35]. Two-dimensional CE with different separation modes has also been demonstrated for proteomics application [36]. Such a 2-D separation scheme employs two columns or capillaries.…”

High sensitive protein detection by hollow fiber membrane interface based protein enrichment and in situ fluorescence derivatization

“…As one of the most sensitive techniques, fluorescence detection is widely used in protein research [7][8][9][10][11]. Although native fluorescence emitted by aromatic amino acid residues (such as ଝ This paper is part of the special issue "Enhancement of Analysis by Analytical Derivatization", Jack Rosenfeld (Guest Editor).…”