【作者】 高明霞；

【导师】 张祥民；

【作者基本信息】 复旦大学 ， 分析化学， 2006， 博士

【摘要】 20世纪中期以来，随着DNA双螺旋结构的提出和蛋白质空间结构的X射线解析，开始了分子生物学时代，对遗传信息载体DNA和生命功能的主要体现者蛋白质的研究，成为生命科学研究的主要内容。90年代中期，在人类基因组计划(Human Genomic Project)研究发展及功能基因组学的基础上，国际上萌发产生了一门在整体水平上研究细胞内蛋白质的组成及其活动规律的新兴学科——蛋白质组学(Proteomics)，它以蛋白质组(Proteome)为研究对象。蛋白质组学(proteomics)已经成为功能基因组学研究的重要领域，其重要性和战略意义日益显著。蛋白质组学研究主要有两种路线：第一种是传统的双向凝胶电泳(2-DE)分离技术。但是双向凝胶电泳技术仍然存在许多不足和一些难以克服的缺陷，比如有限的动态范围，分离样品中分子量差别较大的蛋白、低拷贝蛋白、疏水蛋白以及极酸极碱蛋白的分辨能力比较差，而且其操作过程费时费力，自动化程度低。针对这些固有的技术上的缺陷，具有快速，高效，自动化程度高等优点的液相色谱技术得到了长足发展，并成为蛋白质组学研究的第二条技术路线。发展两维乃至多维色谱分离技术，提高峰容量和分离能力是根本上解决复杂样品分离问题的方法。从理论上说，只要组合在一起的两种模式的分离机理正交，整个二维系统的峰容量是每一维峰容量的乘积，特别适合于复杂样品的分析。在过去的十几年中，已经出现了一些被认为是突破性的进展，Jorgenson(多维色谱-电泳技术)，Yates(Shot-gun技术)，Aebersold(ICAT技术)等科学家在蛋白质组学新方法的研究方面打开了一个崭新的局面。目前，蛋白质组学面临的两大挑战是高丰度蛋白质压制低丰度蛋白质造成低丰度蛋白质检测困难以及蛋白质的定量问题。Shot-gun为代表的bottom-up技术在分离之前将蛋白质样品进行酶解，一个蛋白质酶解成多个肽段，这势必使原本就很复杂的样品变得更加复杂，不仅给色谱的分离能力与质谱的扫描速度提出挑战，不利于低丰度蛋白质的检测，而且将蛋白质酶解成肽段的同时又损失了完整蛋白质的信息。ICAT技术在蛋白质的相对定量方面发挥了重大作用，然而已有的定量技术仅局限在相对定量方面。因此，现有的多维色谱技术在解决蛋白质组学的两大问题上存在许多不足，本论文就是围绕当前蛋白质组学研究中存在的问题开展工作的，建立发展多维色谱技术方法达到实现高丰度蛋白质的去除以及完整蛋白质的分离鉴定以及定量等目的。本论文共分五章。主要内容摘要如下：第一章总结了蛋白质组学的现状与发展以及多维色谱与其相关技术在蛋白质组学中的应用与进展。介绍了二维／多维分离方法的理论依据，探讨了目前蛋白质组学研究使用的技术模式的成功与不足。并介绍了本论文选题的目的和意义，着重在于如何利用多维液相色谱的技术实现在完整蛋白水平上的分离，去除高丰度蛋白质以及利用多维液相色谱技术同时实现对蛋白质的定性定量等问题。第二章以鼠肝为研究对象比较了不同的提取环境对动物组织中蛋白质提取的影响。主要研究了三种提取环境：酸性、中性和碱性环境。利用双向凝胶电泳分离成像技术比较了在三种不同环境下提取的蛋白质的分布的差异性，通过实验证明了在不同的提取环境下提取的蛋白质在种类和含量上都是不同的，同时也证明了在蛋白质组学的研究中不同的分离路线或分离模式应该根据具体分离条件的酸碱度的不同选用不同的蛋白提取方法，以避免在分离的过程中因沉淀问题而损失蛋白质，为后续工作打下基础。第三章针对肝脏组织蛋白建立了反相高效液相色谱／双向凝胶电泳(RPLC／2-DE)的三维分离系统。2-DE技术是比较成熟的蛋白质分离手段，但是样品负载量低是影响中低丰度蛋白检测的一个重要原因，同时2-DE技术对于复杂的蛋白质样品的分离能力是有限的。RPLC是生物分析中常用的高效的分离模式，对蛋白或肽等有很好的分离能力，还具有除盐纯化样品的能力。RPLC与2-DE的分离机理正交，两种技术联合使用能在很大程度上提高样品的分辨率。本章工作以大鼠的肝脏组织蛋白为研究对象建立了实验路线并确立了实验条件。研究表明，蛋白质样品经过RPLC预分离之后，不同的蛋白就被富集浓缩到不同的馏份中，极大的提高了单个馏份中的蛋白在2-DE上的上样量和分辨率，这一点在凝胶扫描图上可以直观的反映出来，同时，实验证明RPLC分离蛋白质有着很好的重现性，据此可以对同一样品进行平行收集，以达到进一步增大单个馏份中蛋白的上样量的目的。因此，将RPLC作为预分离手段用于2-DE之前对样品进行预分离可以提高蛋白质在2-DE上的上样量和分辨率。这条技术路线已经在中国人肝项目表达谱的研究中得到应用。第四章直接采用多维液相色谱的方法通过多维色谱分离的手段去除生物样品中的高丰度蛋白。中心思路就是：首先利用多维色谱技术对蛋白样品进行有效分离，在完整蛋白分离的基础上，将高丰度蛋白质在酶解成更复杂的肽段之前除掉。这条技术路线目前还没有类似报道。具体路线简述如下：发展了强阳离子交换色谱／反相高效液相色谱(SCX／RPLC)分离系统，对鼠肝组织蛋白进行高效的分离。使用连续地线性盐梯度将蛋白质从SCX色谱柱上洗脱下来，得到62个馏份，每个馏份进一步使用常规的RPLC进行第二维地分离，整个分离过程使用紫外信号对蛋白的色谱峰进行监测，在第二维的分离过程中将信号强也就是量大的高丰度蛋白单独收集，其余中低丰度的蛋白进行合并、浓缩和酶解，然后使用毛细管反相色谱(cRPLC)进行分离，之后进行质谱鉴定，62个馏份分别如法炮制。结果表明，一次实验流程可以去除77个高丰度蛋白，占总蛋白量的34.5％，与不经过去除高丰度蛋白的样品进行比较，鉴定蛋白的数量提高了2到3倍之多。在此基础上，我们进一步简化实验路线并提高方法地通量。来自SCX分离之后的62个馏份经过RPLC进一步分离之后，我们将所有含有中低丰度蛋白的洗脱液合并浓缩到一起，共同酶解之后使用在线的二维分离系统，即shot-gun技术对酶解之后的肽段进行分析鉴定。简化后的实验路线对中国人肝样品进行了分析，58个高丰度蛋白质被去除，共鉴定出1213个蛋白质。总之，这套系统对降低蛋白质样品地复杂性，避免高丰度蛋白质在分离和质谱鉴定过程中压制中低丰度蛋白提供了一个新的手段和思路。另外，通过多维液相色谱分离的方法去除高丰度蛋白质，相对通常使用的免疫亲和的手段，无疑更具有通用性、灵活性、低成本和周期短的优势。第五章发展了多维液相色谱的Top-down技术路线，即SCX／cRPLC／靶上酶解／MALDI-TOF-TOF分离鉴定系统。采用SCX／cRPLC分离系统，实现了在完整蛋白水平上的两维分离，本实验室发展的新方法靶上酶解技术的采用将蛋白质分离与质谱鉴定有效的结合起来，从而实现了蛋白从分离、酶解到鉴定这样一个完整的Top-down路线。蛋白质样品经过常规柱SCX分离之后，得到62个馏份，每个馏份继续使用毛细管反相柱cRPLC进行第二维分离，cRPLC作为第二维具有除盐和纯化样品的作用，为直接酶解提供了可能，利用自动点样仪将经过cRPLC的洗脱液直接收集到标准的MALDI靶板上，点样频率为30秒／个，经过靶上单点酶解之后用MALDI-TOF-TOF鉴定。整个系统对中国人肝蛋白进行了鉴定，共鉴定出3313个唯一蛋白。与通常采用的shot-gun技术相比，我们发展的多维液相色谱的Top-down技术路线更能充分的分离蛋白质，得到更多的包括肽谱和疏水性等有关蛋白质的信息。与2-DE相比具有成本低和自动化高等优势。在此基础上，我们进一步发展为三维蛋白分离系统，即SEC／SCX／RPLC系统，对肝脏蛋白进行更为完全的分离，利用紫外可见检测对蛋白峰进行归一化处理，进一步计算出蛋白的含量，相对应的蛋白质色谱峰同时进行浓缩、酶解、进而MALDI-TOF-TOF检测定性分析，以达到对单个蛋白同时进行定性和定量的目的。这条路线已经进行了有成效的探索，得到一些初步的结果，为蛋白质组学中的定量问题提供了一个可行的有价值的新思路。总之，本论文围绕蛋白质组学的研究热点，以液相色谱分离系统为基础，发展了多种有效的分离系统，以完整蛋白质的分离为指导思想，建立了反相色谱预分离结合双向凝胶电泳的分离路线；开创了基于多维色谱分离为基础的高丰度蛋白质去除系统；发展了多维液相色谱的Top-down技术路线，试图在蛋白质研究主流的2-DE-MS技术之外，另辟蹊径，对蛋白组学新方法学的建立进行了有益的探索和研究，而且多数实验路线已经在中国人肝项目中投入使用，贡献了大量的有价值的实验数据。更多还原

【Abstract】 Since 1950’s, scientific interest has been increasingly shifting to molecular biology with the accomplishment of the gene sequencing of Human Genome Project (HGP). Proteomics has been the focus due to the role of executor of the life function. Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. The investigation of functional proteome is of great significance given that functions of most of genes are still not well understood. Presently proteomic research focuses on differential proteome analysis of human diseases, which is designed to find key proteins with potentials to be used as markers for diagnosis or targets for medication. Proteomics is also becoming the spotlight of analytical chemistry.2D PAGE is the most universal method for proteomics and posses high resolve power for proteins. Some problems in 2D PAGE, however, are still difficult to be overcome such as limited pH range, time-consuming and inefficient sensitivity for low abundant proteins. The reality has motivated considerable effort in developing new technique to resolve proteomics as alternative approach to 2D PAGE.Liquid chromatography technologies are powerful technology to resolve proteins. But conventional one dimensional LC systems are incapable of completely resolving all the components in a complex mixture due to insufficient peak capacity. The development of two-or multi-dimensional column based separation techniques greatly improve the resolve power and peak capacity. During the past decade, a lot of analysts contributed to this field, such as Jorgenson’s multi-dimensional separation, Yates’ shot-gun technique, Aebersold’s ICAT technique and so on.This dissertation is divided into 6 parts.In the first chapter, advances in 2D and multidimensional separation techniques were summarized with details. The theory basis of multidimensional separation techniques was introduced and the advantages and shortcomings of existing technical modes were discussed. The intention and meaning of this dissertation were explained.In chapter 2, we used rat as animal model to compare the effect of different extraction conditions on the extracted proteins. The extract methods of tissue sample at acidic, neutral and basic pH were described and compared by using two-dimensional gel electrophoresis and reversed-phase liquid chromatography. The images on gel and mass spectra showed that thedifferent method can enrich proteins at different pH range. Different pH extract methods can be applied into corresponding liquid chromatography mode with the compatible mobile phase so that the loss and precipitation could be avoided. The good reproducibility enables the sample preparation methods to be widely applied in proteomics analysis.In chapter 3, we describe an approach for fractionating complex protein samples from rat liver prior to 2-DE using RPLC. The study indicated that RPLC prefractionation can provide strong enrichment effect which enabled us to visualize additional and less abundance proteins. Chromatographic enrichment was also demonstrated by the peptide mass fingerprint data, which gave mass spectra with increased number of detected peptide and improved signal intensity. The reproducibility of this prefractionation technology allows pooling of several consecutive runs of the same sample, resulting in a highly enrichment of low abundance proteins. This technology route has been applied into the study of profiling mode of Chinese Human Liver Proteomics Project (CHLPP).In chapter 4, for the first time a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of immuno-affinity chromatography, was developed at the intact protein level for depletion of high abundance proteins from rat liver. The core idea was to deplete the high abundance proteins before the digestion.Proteins bound to the SCX phase were eluted off using a consecutive linear salt gradient. After salt fractionation, 62 fractions were obtained. Every fraction was separated further by RPLC. Ultraviolet signal was used to detect the separation process. The protein peaks with signal intensity above 0.1 AU was defined as high abundance proteins, whereas the other was defined as middle- or low high abundance protein. Then, the effluents contained high abundance proteins were collected individually and other effluents were pooled together. The combined middle- or low high abundance proteins were lyophilized, digested and separated by capillary reversed phase liquid chromatography (cRPLC). Mass spectrometry was used to analyze the protein. 62 fractions were treated using the same method. The results were that 77 high abundance proteins were depleted in an experimental process and identified proteins number was increased 2-3 times.Based on the results, a simplified rapid-speed and high-throughput protocol was put forward further. Instead of separating every fraction using cRPLC, all effluents containingmiddle- or low abundance protein were combined, enriched, digested and separated by online shotgun technology. The simplified method was used to analyze Chinese liver sample. 58 high abundance proteins were depleted and 1213 proteins were identified.In summery, the system presented a novel idea about the depletion of high abundance proteins. In contrast with affinity technology, this strategy with the advantages of low-cost and saving-time has no bias to any proteins and could realize the global proteins analysis.In chapter 5, for the first time, the 2-D separation platform based on the liquid chromatography combined with on-probe digestion technique and identification of mass spectrometry was constructed in intact proteins level for separation and identification of complex liver tissue proteins. The 2-D liquid chromatographic system was constructed by coupling SCX with cRPLC. Proteins were prefractionated by SCX according to charge in the first dimensional separation. 62 fractions eluting from the first dimension were subjected automatically to cRPLC to separate similarly charged proteins on the basis of their various hydrophobicities. A novel rapid on-target digestion technique was adopted to couple effectively the separation technique and mass spectrometry technique. After digestion, the matrix was deposited on the same spot, respectively. Then, the dried plate was analyzed by mass spectrometry. This new technology coupled the top-down approach and bottom-up technologies. The high efficiency of system was demonstrated for analysis of intact proteins from the soluble lysates of normal human liver. A total of 3313 proteins were identified.We further developed three-dimensional separation system combined SEC with the SCX/RPLC mentioned above. This 3D system exhibited the potential of further high resolution for extremely complicated mixture. The intention of 3D system was to realize the identification and quantification of proteins. We had explored some relative experiments and made some effective results.In a word, the main contributes of this dissertation is the development of effective separation system based multi-dimensional system. We aim at exploring and finding out new technological systems for proteomics, so that more breakthroughs can be obtained at the qualitative analysis and quantification study.更多还原