Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues
Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues
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DOI：
10.1021/ac061029
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发表时间：
2006-09-01
影响因子：
7.4
通讯作者：
Dovichi, Norman J.
中科院分区：
文献类型：
--
作者：
Kraly, James R.;Jones, Megan R.;Dovichi, Norman J.
我们构建了一个高速、二维毛细管电泳系统，并配备了一个紧凑、高灵敏度的荧光检测器。该仪器用于快速和可重复地分离Barrett食管组织匀浆。蛋白质和生物胺用荧光试剂3-（2-呋喃甲酰基）喹啉-2-甲醛标记。标记的生物分子在两个毛细管中顺序分离。第一毛细管采用使用可替换筛分基质的毛细管筛分电泳。馏分依次转移到第二个毛细管，在那里它们通过胶束电动毛细管色谱进行额外的分离。全面的二维分离需要60分钟。一天内的迁移时间再现性优于1%，在两个维度的50个最强烈的功能。CSE的日间迁移时间精度为1.3%，MECC的日间迁移时间精度优于0.6%。在知情同意的情况下，从近端管状食管的鳞状上皮、远端食管的Barrett上皮和3例Barrett食管患者的胃底区域获得活检。我们确定了18个功能的匀浆配置文件作为生物胺和氨基酸。对于每一个患者，巴雷特的活检有超过5倍的苯丙氨酸和丙氨酸水平相比，鳞状组织。在所有组织类型中，高度异型增生患者的13种氨基酸浓度最高。高度异型增生患者鳞状细胞活检组织中甘氨酸浓度比Barrett's和胃底活检组织高40倍。这些结果表明，二维毛细管电泳可能是有价值的内镜和手术活检的快速表征。
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.