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首页> 外文期刊>Analytical chemistry >Development of non-gel-based two-dimensional separation of intact proteins by an on-line hyphenation of capillary Isoelectric focusing and hollow fiber flow field-flow fractionation
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Development of non-gel-based two-dimensional separation of intact proteins by an on-line hyphenation of capillary Isoelectric focusing and hollow fiber flow field-flow fractionation

机译：通过毛细管等电聚焦在线连接和中空纤维流场-流分馏技术在线开发完整蛋白质的非凝胶二维分离技术

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摘要

A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (FlFFF). The method developed here (CIEF-HFFlFFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFFlFFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.
机译：一种快速的，非基于凝胶的，在线二维分离方法被用于蛋白质组分析。首先通过利用等电聚焦（IEF）利用特氟龙毛细管中它们各自的等电点（pI）的差异来进行蛋白质分离，然后通过流场-流动分离在中空纤维中进行基于分子量（MW）的分离（FlFFF）。此处开发的方法（CIEF-HFFlFFF）可能是二维聚丙烯酰胺凝胶电泳的有力替代方法，该技术目前用于蛋白质的分离和纯化。在CIEF-HFF1FFF中，蛋白质可以一定pI和MW间隔的一部分收集而不会被变性。另外，在中空纤维的分离过程中同时去除了两性溶液，并且总的处理时间显着减少。此方法应用于人类尿液蛋白质组样品，从而在对每个收集到的蛋白质组分进行胰蛋白酶消化后，鉴定了114种蛋白质，随后脱机使用了纳流液相色谱-串联质谱法。

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