功能化磁性纳米材料的制备及其在蛋白质组学中的应用方法研究

发布时间：2018-01-02 19:35

本文关键词：功能化磁性纳米材料的制备及其在蛋白质组学中的应用方法研究　出处：《北京工业大学》2016年博士论文　论文类型：学位论文

【摘要】：随着生命科学研究飞速发展和后基因组学时代到来,蛋白质作为生命活动的执行体,调节着生物体内诸多重要的生理生化活动。监测蛋白质动态变化、翻译后修饰状态以及研究蛋白质结构功能等对于人们认识生命活动的本质有着极为重要意义。其中,蛋白质磷酸化不仅参与生物体内诸多生命活动,还与多种疾病的发生发展息息相关。作为蛋白质组学研究的核心技术,生物质谱在磷酸化蛋白质组研究中发挥着重要作用并得到了广泛应用。然而,由于生物样本的复杂性以及在磷酸化蛋白质组分析中存在的大量非磷酸化肽段对磷酸肽质谱分析造成的信号干扰,使磷酸化蛋白质组分析面临巨大挑战。此外,在“bottom-up”分析策略中,传统溶液酶切法耗时长,易发生酶的自切,极大影响了蛋白质鉴定通量和覆盖率。因此,发展快速、高效的蛋白质样品预处理新方法是提高蛋白质分析灵敏度和鉴定效率的关键。本文针对以上问题,(1)发展了两种基于新型功能化磁性纳米材料的磷酸肽富集分离新方法并成功应用于实际生物样本,克服了基于固相金属离子亲和色谱法富集磷酸肽特异性差的缺点,并提高了磷酸肽的鉴定效率;(2)制备了一种基于磁性金属有机框架(metal-organic frameworks,MOFs)材料的新型固定化酶反应器,显著提高了蛋白质酶切效率并成功应用到小鼠卵母细胞蛋白样本的快速高效酶切。本论文中,首先阐述了不同材料在蛋白质组学样品前处理过程中的国内外研究现状,包括功能化磁性纳米材料用于磷酸肽的快速富集和固定化酶反应器,以及金属有机框架材料的研究进展与应用等。其次,基于镧系金属表面电子云分布及其与磷酸根可产生相互作用的特征设计合成了一种混合镧系金属(Tb~(3+),Tm~(3+),Ho~(3+),Lu~(3+))固载的新型功能化磁性纳米材料,Fe_3O_4@TCPP-DOTA-M~(3+),并将其应用于磷酸肽的快速、特异性富集。材料合成中,TCPP(tetrakis(4-carboxylphenyl)porphyrin)的应用不仅可以提高DOTA和镧系金属离子的负载量进而增加材料与磷酸肽的碰撞几率,同时也可以提高磁性纳米材料的亲水性从而获得更高的磷酸肽富集效率。另外,大环配合物DOTA(1,4,7,10-tetraazacyclododecane N,N’,N’’,N’’’-tetraacetic acid)可与镧系金属螯合形成热稳定性极高的金属复合物,进一步增强材料的整体稳定性。实验结果表明,当5 pmolα-酪蛋白酶切产物经新材料富集分离后可鉴定19条磷酸肽。即使在100倍(摩尔比)BSA蛋白酶切产物干扰下,经新材料富集分离后仍可鉴定到16条磷酸肽,其特异性明显优于商品化的TiO2材料的富集鉴定结果。该新型功能化磁性纳米材料进一步应用于HeLa细胞蛋白提取样品的磷酸肽富集。在单次质谱鉴定后,可成功检测到9048条磷酸化肽段对应2103个磷酸化蛋白质组。另外,为了系统分析不同金属离子与磷酸根相互作用行为差异,设计合成了不同金属离子(Ti~(4+),Zr~(4+),Fe~(3+),Tb~(3+),Tm~(3+),Ho~(3+))固载的新型功能化磁性纳米材料,Fe_3O_4@TCPP-DOTA-Ms,并成功应用于多位点磷酸化肽段特异性富集。实验结果表明,Fe_3O_4@TCPP-DOTA-Tb,Ti富集磷酸肽效果明显优于其他金属离子固载的材料,并且在鉴定到的磷酸肽中多位点磷酸化肽段的比例也较高。实验结果进一步证明,固载过渡金属的磁性纳米材料富集磷酸肽的灵敏度优于固载镧系金属的材料,而固载镧系金属的磁性纳米材料富集磷酸肽的选择性优于固载过渡金属的材料。结合过渡金属和镧系金属的优势,采取Fe_3O_4@TCPP-DOTA-Tb/Ti联合富集的策略,在HeLa细胞中成功鉴定到了13450条磷酸肽对应2965个磷酸化蛋白质组,富集特异性高达94%。其中,多位点磷酸化肽段占总磷酸肽的比例超过了50%,结果明显优于DHB/TiO2的富集鉴定方法(13.39%)。最后,由于固定化酶反应器可极大缩短蛋白质酶切时间,且MOFs材料具有较大的比表面积和可选择的作用位点,因此将二者结合制备了一种磁性MOFs材料基固定化酶反应器,Fe_3O_4@DOTA-ZIF-90-trypsin,并成功应用于小鼠卵母细胞蛋白样本的蛋白质组分析。本工作合成的新型磁性MOFs材料既具有良好的磁响应,又具备MOFs材料比表面积大、结构及热稳定性高等优势。将胰蛋白酶通过与磁性MOFs材料表面的大量醛基共价连接在磁性MOFs材料表面,获得新型固定化酶反应器。实验结果表明,BSA蛋白经1 min酶切后,获得了77%的氨基酸序列覆盖率,其酶切效率优于12 h传统溶液酶切结果(70%)。小鼠卵母细胞蛋白提取样本经20 min酶切后,单次质谱鉴定可检测到8957条肽段对应1843个蛋白质组。与传统溶液酶切相比,在酶切后鉴定的肽段和蛋白质组数目上新方法有67%和40%的提高。本研究不仅发展了一种磁性MOFs材料快速有效的合成策略,同时也进一步扩大了MOFs材料在蛋白质组学中的应用。
[Abstract]:With the rapid development of life science research and the post genomic era, as the executive body protein of life activity, regulate organism many important physiological activities. The dynamic changes of protein post-translational modification status monitoring, and research in protein structure and function in essential understanding of life activities is of great significance. Among them, protein phosphorylation is not only involved in biological life activities, but also with a variety of diseases is closely related to the development. As the core technology of proteomics research, biological mass spectrometry of proteins plays an important role in the study and is widely used in phosphoric acid. However, due to the complexity of biological samples as well as in the existing phosphorylation proteome analysis in a large number of non phosphopeptide analysis of signal interference caused by the peptide mass of phosphoric acid, the phosphoproteome analysis Facing a huge challenge. In addition, in the "bottom-up" analysis method, the traditional solution of enzyme digestion method is time-consuming and prone to self cutting enzyme, which greatly affects the flux and protein identification coverage. Therefore, the rapid development of the new method, the protein sample pretreatment is the key to improve the efficiency of protein analysis sensitivity and identification efficiency. Aiming at the above problem, (1) the development of two kinds of magnetic nano materials based on the new function of phosphopeptide enrichment and separation method and applied to biological samples, immobilized metal ion affinity chromatography overcomes the shortcomings of the poor enrichment of phosphorus acid peptide specific based on and improve the identification efficiency of phosphopeptides (2;) to prepare a magnetic metal organic framework based on (metal-organic frameworks, MOFs) model of immobilized enzyme reactor materials, significantly increased the protein digestion efficiency and successfully applied to protein mouse oocytes Fast and efficient enzyme sample cut. In this thesis, first elaborated the domestic research status of different materials in the proteomics sample pretreatment process, including functional magnetic nano materials for rapid enrichment and immobilized enzyme reactor phosphopeptide, and the study of metal organic framework materials development and application. Secondly, lanthanide metal surface electron cloud distribution and phosphate can produce a mixture of lanthanide metal synthesis feature design based on interaction (Tb~ (3+), Tm~ (3+), Ho~ (3+), Lu~ (3+)) supported the new functional magnetic nano materials, Fe_3O_4@TCPP-DOTA-M~ (3+). And its application in phosphopeptide enrichment. The rapid, specific materials in the synthesis of TCPP (tetrakis (4-carboxylphenyl) porphyrin) the application can not only improve the loading amount of DOTA and lanthanide metal ions and increase the collision probability of materials and phosphopeptides, at the same time In order to improve the hydrophilicity of magnetic nano materials to obtain higher efficiency of phosphopeptide enrichment. In addition, the macrocyclic complexes of DOTA (1,4,7,10-tetraazacyclododecane N, N ', N', N '' -tetraacetic acid ') and lanthanide metal chelate forming high thermal stability of the metal complex, and further enhance the overall stability of the material the experimental results show that when 5 pmol of alpha casein digestion products by new material enrichment and separation after identification of 19 phosphopeptides. Even in 100 times (molar ratio) BSA protease digestion products by interference, new materials can be identified by the enrichment and separation of 16 phosphopeptide enrichment and identification results, the TiO2 material the opposite sex is obviously better than that of commercialization. The new functionalized magnetic nano material is further applied to HeLa cell protein extraction of phosphopeptide enrichment. Samples in a single mass spectrometry after successfully detected the 9048 phosphopeptides corresponding to 21 03 protein phosphorylation. In addition, in order to analyze the different metal ions and phosphate interaction behavior differences, different metal ions were designed and synthesized (Ti~ (4+), Zr~ (4+), Fe~ (3+), Tb~ (3+), Tm~ (3+), Ho~ (3+)) new function supported the magnetic nano materials, Fe_3O_4@TCPP-DOTA-Ms, and successfully applied to multilocus phosphopeptide specific enrichment. The experimental results show that Fe_3O_4@TCPP-DOTA-Tb, Ti enrichment of phosphopeptides is superior to other metal ion immobilized materials, and to the identification of phosphopeptides in multi site phosphorylated peptide proportion is higher. The experimental results further evidence that the sensitivity is better than solid supported transition metal nanometer magnetic materials for enrichment of phosphopeptides immobilized lanthanide metal materials, the selectivity is better than immobilized lanthanide metal magnetic nano material enrichment of phosphopeptides supported transition metals with transition metal and materials. Take advantage of the lanthanide metal, Fe_3O_4@TCPP-DOTA-Tb/Ti enrichment strategy, identified 13450 phosphopeptides corresponding to 2965 protein phosphorylation in HeLa cells, specific enrichment of up to 94%. the multilocus phosphopeptides total phosphopeptide ratio of more than 50%, the result is better than that of DHB/TiO2 enrichment and identification methods (13.39%). Finally, the immobilized enzyme reactor can greatly shorten the protein digestion time, and MOFs material has larger surface area and alternative sites of action, so a kind of magnetic material based MOFs immobilized enzyme reactor was prepared by the combination of the two Fe_3O_4@DOTA-ZIF-90-trypsin, and proteomic analysis the successful application in mouse oocyte protein samples. In this work, the synthesis of novel magnetic MOFs material has good magnetic response, but also have MOFs large surface area, high thermal stability and structure The advantages of trypsin. With large aldehyde surface magnetic MOFs material connected covalently on the surface of the magnetic material MOFs, novel immobilized enzyme reactor. The experimental results show that the BSA protein was 1 min after enzyme digestion, the amino acid sequence of 77% coverage, the digestion efficiency is superior to the traditional 12 h solution the results of restriction (70%). The protein of mouse oocyte samples were extracted by 20 min enzyme digestion, single mass spectrometry can detect 8957 peptides corresponding to 1843 proteins. Compared with the conventional in solution digestion, digestion identification number of peptides and proteins were new methods and 67% 40% improved. This study not only the development of the synthetic strategy of a magnetic material MOFs quickly and effectively, but also to further expand the MOFs material and its application in proteomics.

【学位授予单位】：北京工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51;TB383.1

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