Liquid-based free-flow electrophoresis–reversed-phase HPLC: a proteomic tool
Liquid-based free-flow electrophoresis–reversed-phase HPLC: a proteomic tool
复制标题
液基自由流动电泳-反相 HPLC：蛋白质组学工具
作者：
R. Moritz;R. Simpson
基于MS的复杂蛋白质混合物蛋白质组学分析的核心问题之一是蛋白质丰度的大动态范围问题，细胞的蛋白质丰度可能在105到106之间，血液等组织的丰度可能在109到1010之间。为了克服这一僵局，基于电泳法、层析法或正交电泳色谱原理，已经开发了许多用于简化复杂蛋白质混合物的预分离方法1，2。本协议描述了一种采用基于液体等电聚焦(IEF)蛋白质分离方法、第一维自由流动电泳3(FFE)和第二维每个FFE池的快速离线反相高效液相色谱(RP-HPLC4)的正交分离过程。在FFE步骤中，复杂的蛋白质混合物被连续地注入到载体两性离子溶液中，在两个平行板之间以薄层状薄膜(0.4-1.0 mm)流动(图1)。通过引入垂直于流动方向的电场，IEF根据蛋白质的等电点值分离蛋白质，并将其收集到96个明确的池中，每个池以~0.02-0.10pH单位分隔。PH梯度的性质可以通过明智地选择两性离子来定制。通过在分离缓冲液中加入共沸剂(如6M尿素)和还原剂(如二硫苏糖醇(DTT))，可以在非变性或变性条件下进行离子交换。然后，对每个FFE池进行分析(例如，FFE池的~2.5%)或制备快速反相高效液相色谱法(每次分析~1-6分钟)。
One of the central problems of mass spectrometry (MS)-based proteomic analysis of complex protein mixtures is the issue of the wide dynamic range of protein abundances, which may vary from 105 to 106 for cells and 109 to 1010 for tissues such as blood. To overcome this impasse, many prefractionation methods for simplifying complex protein mixtures, based on either electrophoretic, chromatographic or orthogonal electropheretic-chromatographic principles, have been developed1,2. This protocol describes an orthogonal separation procedure utilizing a liquid-based isoelectric focusing (IEF) protein separation method, free-flow electrophoresis3 (FFE) in the first dimension and rapid off-line reversed-phase highperformance liquid chromatography4,5 (RP-HPLC) of each FFE pool in the second dimension. In the FFE step, complex protein mixtures are continuously injected into a carrier ampholyte solution flowing as a thin laminar film (0.4–1.0 mm) between two parallel plates (Fig. 1). With the introduction of an electric field perpendicular to the direction of flow, proteins are separated by IEF according to their pI values and collected into 96 well–defined pools, each separated by ~0.02–0.10 pH unit. The nature of the pH gradient can be customized by the judicious choice of ampholytes5. IEF can be performed in either nondenaturing or denaturing conditions by the addition of a chaotropic agent (for example, 6 M urea) and a reducing agent (for example, dithiothreitol (DTT)) in the separating buffer. Each FFE pool is then subjected to either analytical (for example, ~2.5% of FFE pool) or preparative rapid RP-HPLC (~1–6 min per either analysis).
影响因子：
2.9
作者：
EGEN, NB;BLISS, M;BIER, M
通讯作者：
BIER, M