Microfabricated two-dimensional electrophoresis device for differential protein expression profiling
Microfabricated two-dimensional electrophoresis device for differential protein expression profiling
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DOI：
10.1021/ac0711485
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发表时间：
2007-10-01
影响因子：
7.4
通讯作者：
Mathies, Richard A.
中科院分区：
文献类型：
--
作者：
Emrich, Charles A.;Medintz, Igor L.;Mathies, Richard A.
建立了一种微流控分离系统，用于对大肠杆菌诱导蛋白表达产生的复杂细胞蛋白混合物进行二维差异凝胶电泳（DIGE）分离。杆菌微型DIGE分析仪是一种双层硼硅酸盐玻璃微型装置，由一个3.75 cm长的等电聚焦通道组成，通过20个通道平行采样，通过自然电泳实现二维分离。正交分离系统之间的连接通过包含微流体界面（MFI）的较小通道来实现，该微流体界面防止两个维度之间的介质泄漏，并且使得能够在每个维度上容易地加载不连续的凝胶系统。用Cy2和Cy3 DIGE共价标记蛋白质，并用旋转共聚焦荧光扫描仪同时检测。纯化蛋白质和复杂蛋白质混合物的可再现二维分离通过包括7 M尿素以最小的运行间变化进行。在第二维分离矩阵中。通过跟踪麦芽糖结合蛋白在大肠杆菌中的诱导表达，证明了微型DIGE分析仪的能力。杆菌虽然十二烷基硫酸钠（SDS）在第二维尺寸分离的情况下限制了分离的正交性和峰值容量，但该分析仪是朝着在微加工设备中对复杂蛋白质样品进行可重现的二维分析迈出的重要的第一步。此外，这里开发的微通道接口结构将促进微器件中的其他多维分离。
A microfluidic separation system is developed to perform two-dimensional differential gel electrophoretic (DIGE) separations of complex, cellular protein mixtures produced by induced protein expression in E. coli. The micro-DIGE analyzer is a two-layer borosilicate glass microdevice consisting of a single 3.75 cm long channel for isoelectric focusing, which is sampled in parallel by 20 channels effecting a second-dimension separation by native electrophoresis. The connection between the orthogonal separation systems is accomplished by smaller channels comprising a microfluidic interface (MFI) that prevents media leakage between the two dimensions and enables facile loading of discontinuous gel systems in each dimension. Proteins are covalently labeled with Cy2 and Cy3 DIGE and detected simultaneously with a rotary confocal fluorescence scanner. Reproducible two-dimensional separations of both purified proteins and complex protein mixtures are performed with minimal run-to-run variation by including 7 M urea. in the second-dimension separation matrix. The capabilities of the micro-DIGE analyzer are demonstrated by following the induced expression of maltose binding protein in E. coli. Although the absence of sodium dodecyl sulfate (SDS) in the second-dimension sizing separation limits the orthogonality and peak capacity of the separation, this analyzer is a significant first step toward the reproducible two-dimensional analysis of complex protein samples in microfabricated devices. Furthermore, the microchannel interface structures developed here will facilitate other multidimensional separations in microdevices.