Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome
Multi-component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome
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DOI：
10.1002/pmic.200390057
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发表时间：
2003-04-01
期刊：
影响因子：
3.4
通讯作者：
Steiner, S
中科院分区：
文献类型：
--
作者：
Pieper, R;Su, Q;Steiner, S
为了发现指示疾病过程或药物效应的新的蛋白质标志物，最常用的蛋白质组学技术平台包括双向电泳(2-DE)高分辨率蛋白质分离、从染色的凝胶斑点中鉴定蛋白质的质谱学以及由统计学支持的生物信息学数据分析过程。这种方法在分析组织匀浆中的蛋白质及其与疾病或治疗相关的定量变化方面比在血浆样本中更成功。血浆蛋白质的显示和定量有几个缺点：少数蛋白质丰度很高；许多蛋白质的高度异质性导致长的电荷串；2-DE分离的蛋白质斑点拥挤，分子质量在45-80kD之间，等电点在4.5-6之间。因此，需要蛋白质组学技术来解决这些问题，特别是能够准确定量血浆和其他体液中大量不丰富的蛋白质。本文介绍的基于免疫亲和力的蛋白质消减层析(IASC)可以有效和重复性地去除高浓度血浆和血清中存在的多种蛋白质。应用IASC作为2-DE的前期血浆样品制备过程，凝胶中观察到的蛋白质斑点模式发生了显著变化，至少额外显示了350个低丰度蛋白质。亲和纯化多克隆抗体(PABS)是一种免疫亲和试剂，可特异性去除人血浆样品中的白蛋白、免疫球蛋白G、免疫球蛋白A、转铁蛋白、结合珠蛋白、α-1-抗胰蛋白酶、血凝素、转甲状腺蛋白、α-2-HS糖蛋白、α-1-酸性糖蛋白、α-2-巨球蛋白和纤维蛋白原等。为了使免疫亲和力减法过程可循环使用，将PAb固定并交联在层析基质上。每个针对一种蛋白质的抗体偶联基质可以混合在一起形成混合床IASC柱。我们发现，在一个步骤中，多达十个具有相似溶解特性的亲和结合的血浆蛋白可以从混合床柱中洗脱出来。这促进了高通量的血浆样本的自动化层析处理，这是蛋白质组疾病标记物发现项目中所希望的。
In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Anti body-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.