基于免疫亲合分离技术的人血浆蛋白质组研究

发布时间：2018-01-12 06:34

本文关键词：基于免疫亲合分离技术的人血浆蛋白质组研究　出处：《中国人民解放军军事医学科学院》2006年博士论文　论文类型：学位论文

【摘要】：人血液含有来源于几乎所有细胞、组织、器官的蛋白质，可以直接反映病理、生理状态，是各种疾病诊断、生物标志物发现的最有价值的标本。因此，血浆蛋白质组成为人们研究的热点。而血浆蛋白质又具有独特的性质(蛋白质组成、功能及结构形式极端复杂)，高低丰度蛋白质含量的动态范围可差10~(12)之巨，限制了质谱对低丰度蛋白的有效检测，对血浆蛋白质组学的研究技术体系提出了挑战。因此，如何去除血浆中高丰度蛋白质从而使低丰度蛋白质得以富集(降低高丰度蛋白质对低丰度蛋白质鉴定的干扰)，成为血浆蛋白质鉴定的关键和研究热点。目前基于免疫亲合去除血浆高丰度蛋白质的技术得到研究者普遍认可和广泛的应用，但由于血浆中几种高丰度蛋白质是转运蛋白质或结合蛋白，高丰度蛋白质的去除可能会造成其他蛋白质的损失，这是血浆蛋白质组表达谱和差异蛋白质组研究者普遍关注的问题，，也是本课题研究的主要内容；另外，对免疫亲合系统处理样本时造成蛋白质非特异丢失的原因进行了考察与分析；基于两种免疫亲合分离(而非免疫亲合去除)技术的血浆蛋白质组研究，使更大范围的血浆蛋白质鉴定得以实现，为今后大规模血浆蛋白质组研究提供新的思路。在本研究中，我们用目前广泛应用的可同时去除六种高丰度蛋白质的免疫亲合色谱柱(Agilent MARS)和刚刚发展的同时去除十二种高丰度蛋白质的免疫亲合色谱柱(Genway SepproTM MIXED12)分离人血浆样本，分别得到与亲合柱结合的组分(MARS-BF和SepproTM-BF)和流出组分(MARS-FF和SepproTM-FF)。这四个蛋白质组分别经胰蛋白酶酶切后得到复杂肽混合物，采取离线阳离子交换色谱分离及反相色谱离子阱串联质谱鉴定的技术体系，分别对肽混合物进行分离鉴定。采用国际人类蛋白质组组织血浆蛋白质组项目(HUPO PPP)推荐的过滤参数，共鉴定蛋白质2401种，属于较大的血浆蛋白质组数据集。当采用更严格的过滤参数，四个组分共鉴定蛋白质529种，其中277种蛋白质是HUPO PPP鉴定的889种中没有覆盖的。鉴定蛋白质的动态范围为9个数量级(albumin，35mg／mL-Angiotensinogen precursor，＜10pg／mL)。采用搜索反转数据库的方法评价数据可靠性，结果表明肽段鉴定的可靠性从73.6％上升到99％。为了确定结合组分中大量的非靶蛋白质的存在是否是由于其和高丰度蛋白质的相互作用造成的，我们对白蛋白做了免疫共沉淀实验，并对其结合蛋白质进行了质谱
[Abstract]:People with blood from almost all cells, tissues, organs of the protein, can directly reflect the pathological and physiological state, is a variety of disease diagnosis, biomarker discovery of the specimens. Therefore, plasma protein composition is the focus of the research. The plasma protein has unique properties (proteins. The function and structure of the complex dynamic range), extreme abundance protein content difference of 10~ (12) of the giant, limits the effective detection of low abundance protein mass spectrometry, it is a challenge to the technical system of plasma proteomics. Therefore, how to remove high abundance proteins in plasma and enrich the low abundant proteins (to reduce the interference of high abundance proteins of low abundance protein identification), is a key and hot research of plasma protein identification. At present based on immunoaffinity removal of high abundance plasma proteins The researchers generally accepted and widely used, but because of some high abundance proteins in plasma is a transporter protein or binding protein, high abundance protein removal may cause the loss of other proteins, the plasma proteome expression and proteome research issues of common concern, but also the main contents of this research in addition, the; of immunoaffinity sample processing system when the cause of protein loss were studied; two based on immunoaffinity separation (not immunoaffinity removal) plasma proteomic techniques, so that a wider range of plasma protein identification can be realized, for future large-scale plasma proteomics the research provides new ideas.
In this study, we applied the current widely used can simultaneously remove immune six high abundance protein affinity chromatography column (Agilent MARS) and immune development just get rid of twelve high abundance protein affinity chromatography column (Genway SepproTM MIXED12) isolated from human plasma samples obtained with affinity column combination group respectively (MARS-BF and SepproTM-BF) and outflow components (MARS-FF and SepproTM-FF). These four proteins were digested by trypsin complex peptide mixtures, technology system taken offline cation exchange chromatography separation and reversed-phase chromatography ion trap tandem mass spectrometry identification, isolation and identification of peptide mixtures were used. The International Human Proteome Organization plasma proteome project (HUPO PPP) criteria, 2401 proteins were identified, belonging to the plasma proteome. When using larger data sets more stringent The filter parameters, four components were identified 529 proteins, 277 proteins which is HUPO PPP identification is not covered in 889. The dynamic range of the protein is 9 orders of magnitude (albumin, 35mg / mL-Angiotensinogen < precursor, 10pg / mL). The database search inversion method to evaluate the reliability of data and the results show that the reliability of peptides increased from 73.6% to 99%. in order to determine the binding of non target protein components in the presence of a large number of whether it is the result of its interaction with high abundance proteins, we do albumin co immunoprecipitation experiments, and the combination of protein mass spectrometry

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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