Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues - PubMed (original) (raw)

. 2006 Sep 1;78(17):5977-86.

doi: 10.1021/ac061029+.

Megan R Jones, David G Gomez, Jane A Dickerson, Melissa M Harwood, Michael Eggertson, Thomas G Paulson, Carissa A Sanchez, Robert Odze, Ziding Feng, Brian J Reid, Norman J Dovichi

Affiliations

Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues

James R Kraly et al. Anal Chem. 2006.

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.

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Figures

Figure 1

Figure 1

2D-CE interface. Two 140 μm OD × 30 μm ID separation capillaries are inserted into two 365 μm OD × 148 μm ID capillary sleeves and aligned coaxial to each other approximately 40 μm apart.

Figure 2

Figure 2

Voltage timing diagram for 2D-CE experiments. All voltages are applied in negative polarity and the sheath-flow cuvette is held at ground potential. The voltage applied to each capillary is determined by the net potentials between electrode 1, electrode 2, and the detector (ground).

Figure 3

Figure 3

Injection of 3 nM FQ-labeled carbonic anhydrase separated by MEKC. The 3σ limit of detection is 330 yoctomol (10−24 mol).

Figure 4

Figure 4

One-dimensional capillary electrophoresis separations. A) CSE separation of BE cellular homogenate. B) MEKC separation of BE cellular homogenate. C) CSE separation of amino acid standards, peak 4 – glutamic acid, 5 - lysine, 6 - phenylalanine, 7 – isoleucine, 9 – arginine, 10 - methionine, 12 – alanine, 17 - glutamine, 18 - histidine. All separations at -1000 V/cm.

Figure 5

Figure 5

Two-dimensional electrophoresis separation of BE cellular homogenate A) Landscape plot. Fluorescence intensity is plotted on the z-axis. Component intensity spans 4 orders of magnitude. B) Expanding the data 10X illustrates the high sensitivity of laser induced fluorescence detection. C) The same data viewed as a gel image by overexposing the image to show the low amplitude components.

Figure 6

Figure 6

Four 2D-CE landscape images from replicate experiments showing the separation of BE cellular homogenate. The within-day migration time reproducibility is better than 1% for the 50 most intense components.

Figure 7

Figure 7

A) 2D-CE separation of BE cellular homogenate B) Separation of BE cellular homogenate treated with trypsin enzyme. Regions are highlighted to show the disappearance of the protein content with the mixture

Figure 8

Figure 8

Identified biogenic amines in 2D-CE separations of BE homogenate. 1 - spermidine, 2 - putresceine, 3 - cadaverine, 4 – glutamic acid, 5 - lysine, 6 - phenylalanine, 7 – isoleucine, 8 – valine, 9 – arginine, 10 - methionine, 11 – glycine, 12 – alanine, 13 - serine, 14 - threonine, 15 – tryptophan, 16 - asparagine, 17 - glutamine, 18 - histidine.

Figure 9

Figure 9

Endoscopic view of Barrett’s esophagus. A) squamous and B) Barrett’s. The fundus is in the stomach beyond C.

Figure 10

Figure 10

Gel images showing 2D-CE separations of adjacent biopsies of squamous and fundal biopsies from a 64 year old female patient with high grade dysplasia. The position of each biopsy is noted as the distance from the incisors.

Figure 11

Figure 11

Gel images showing 2D-CE separations of Barrett’s, squamous, and fundus tissue homogenates from each of three patients. The position of each biopsy is noted as the distance from the incisors.

Figure 12

Figure 12

Landscape images of the biogenic amine region from Figure 11. Spot labeling from Figure 8.

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