Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column
Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column
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DOI：
10.1016/j.chroma.2008.10.104
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发表时间：
2009-03-27
影响因子：
4.1
通讯作者：
Strancar, Ales
中科院分区：
文献类型：
--
作者：
Urbas, Lidija;Brne, Peter;Strancar, Ales
人血清白蛋白（HSA）和免疫球蛋白G（IgG）占人血浆中所有蛋白质的75%以上。这些高丰度蛋白质阻止了作为各种疾病的潜在标志物的低丰度蛋白质的检测。因此，HSA和IgG的消耗对于进一步的蛋白质组分析是必不可少的。在本文中，我们描述了使用亲和和伪亲和层析的HSA和IgG的选择性耗尽的条件的优化。使用BIA Separations CIM（对流相互作用介质）蛋白G盘去除IgG，使用Mimetic Blue SA A6XL固定相去除HSA。根据峰形选择CIM蛋白G纸片的结合和洗脱缓冲液。测定了动态结合容量。它被证明是依赖于所使用的缓冲系统和独立的流速和IgG的浓度。除IgG标准品的结合能力外，还测定了人血浆中IgG的结合能力。使用人血浆表征Mimetic Blue SA A6 XL色谱柱。耗竭的选择性取决于色谱柱上样的人血浆量。在优化两种载体的条件后，将Mimetic Blue SA A6 XL固定相与CIM蛋白G盘组合，以同时耗尽人血浆样品。设计了能够从20 μ L人血浆中去除IgG和HSA的离心旋转柱。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳和双向凝胶电泳检测耗竭结果。(C)2008 Elsevier B.V.保留所有权利。
Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 mu L of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. (C) 2008 Elsevier B.V. All rights reserved.