【作者】 毛煜；

【导师】 张祥民；

【作者基本信息】 复旦大学 ， 分析化学， 2004， 博士

【摘要】 蛋白组学的研究方法,主要依赖于分离分析技术和检测技术的突破性发展?2D PAGE 是最通用的蛋白组分离方法,它结合了等电聚焦和体积排阻两种分离机理正交的分离方法,对蛋白有很高的分辨能力?然而 2D PAGE 也存在一些难以克服的缺陷,比如有限的动态范围,分离样品中大小差别较大的蛋白?低拷贝蛋白(如信号蛋白和转录因子)?疏水蛋白(膜结合蛋白和跨膜蛋白)以及极酸极碱蛋白时仍然存在许多难以克服的问题,对操作者的技术要求高,难以实现与质谱(MS)的直接联用等?寻找 2DE 的替代技术为全球生物分析工作者所关注? 传统色谱或电泳分离技术由于峰容量有限,对于组分异常复杂的体系如蛋白组,分离能力有很大的局限性?发展两维乃至多维色谱-电泳分离技术,是根本上解决问题的方法?从理论上说,只要组合在一起的两种模式分离机理正交,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)等科学家在蛋白组学新方法的研究方面打开了一个崭新的局面? 本论文以多维色谱-电泳分离方法为主要方向,系统地研究了本工作涉及到的毛细管高效液相色谱(RPLC)?毛细管等电聚焦(CIEF)?离子交换色谱(SCX)等分离方法和激光诱导荧光检测技术(LIF)?阵列分离技术以及全柱成像检测方法(WCID)?首创性地建立了 RPLC/CIEF 二维系统?基于阵列 CIEF 的二维和三维分离系统,设计并制作了受专利保护的用于大数量毛细管阵列的全柱成像激光诱导荧光检测器,将此多维系统应用于酵母蛋白组和肝肿瘤组织蛋白组等样品的分离,得到了令人满意的结果,证明了多维色谱-电泳分离系统超强的分离能力和广阔的应用前景?另外还研制了一台激光诱导荧光检测器,能进行芯片?毛细管的单点或全柱检测以及阵列检测? 本论文共分七章,主要内容如下; 论文第一章总结了二维/多维色谱-电泳分离领域的研究进展?介绍了二维/多维分离方法的理论依据,详述了以 IEF 分离模式为基础的多维分离方法,探讨了多维分离模式的成功与不足,同时总结了毛细管阵列分离和全柱成像检测技术的进展?并介绍了本论文选题的目的和意义,着重在于改进多维分离的通量和检测方面的问题? -VII-<WP=9>复旦大学博士学位论文第二章工作探讨了各种因素对 CIEF 分离结果的影响?主要研究了毛细管内壁涂层?盐浓度?两性电解质浓度?电极液浓度和聚焦时间等实验条件对 CIEF分离结果的影响,并对 CIEF 的内在分离机理进行了初步探索,希望通过实验,能对这种高分辨的分离技术有更深入的了解,以充分发挥其分离效率?为后续工作打基础? 第三章首次建立了 RPLC/CIEF/LIF 二维系统?RPLC 是生物分析中常用的高效的分离模式,对蛋白或肽等又很好的分离能力?CIEF 与 RPLC 分离机理正交,它不仅具有传统等电聚焦的高分辨率,又具有快速和易于自动化的优点?而且样品容量大,进样时往往是整个毛细管注满,与 cRPLC 的流量相匹配,且聚焦的过程中蛋白样品能富集几个数量级,特别适合于第二维分离,能弥补第一维分离时造成的样品稀释,提高分析的灵敏度? 用此二维系统对 BSA 酶解肽段和酵母蛋白进行了有效的分离,峰容量达到了10000 左右?并考察了荧光衍生对 CIEF 的影响,选择了巯基衍生染料而不是常用的氨基衍生染料,结果证明巯基衍生对 CIEF 来说是一种适宜的方式,不会引起 pI 的明显漂移,聚焦效果也不会受到影响,而且还不会损失某些蛋白组分尤其是酸性蛋白? 第四章首创了用于 CIEF 阵列的全柱成像激光诱导荧光检测(WCID-LIF),发展了 RPLC/CIEF 阵列/二维系统,对高转移潜能肝癌组织蛋白组进行了高效的分离检测?并对整个二维分离系统和检测系统申请了专利? 第二维采用 CIEF 阵列,使得分析通量得到很大的提高,弥补了 CIEF 相对于凝胶 IEF 在通量上的不足?而且 RPLC/CIEF 阵列二维系统对第二维的分析速度就没有苛刻的要求,因为第一维的馏分在第二维同时分离,可以充分优化 CIEF的分离条件,而不必受到速度的限制?这样,两个高分辨的分离模式组合起来,不损失任何一维的柱效,整个系统就能具有很高的峰容量? 全柱检测对 CIEF 来说是一种更为高效的检测方式,能避免移动检测过程带来的重现性问题?本论文首创性地发展了用于较高毛细管阵列数的全柱成像检测,光源采用半导体激光器,激光束通过激光化线器化为一条直线,直线扫描过整个毛细管阵列,CCD 相机积分记录下阵列中的荧光信号? 用此二维系统,我们成功分离了高转移鼠肝肿瘤组织(D20)蛋白组,得到了满意结果?系统峰容量达到约 20000?而且还探讨了这一系统在定量蛋白组方面的潜力,因为荧光检测具有较宽的动态线性范围,比较适合与蛋白组样品组分浓度悬殊的特点? VIII- -<WP=10>复旦大学博士学位论文在此基础上,我们进一步提出了 SCX/RPLC/CIEF 阵列/WCID-LIF 三维分离的概念,对低转移鼠肝肿瘤组织(D35)进行了更为完全的分离?因为蛋白组的数目非常巨大,二维分离往往也不能够完全解决问题,因此在二维的基础更多还原

【Abstract】 Scientific interest has been increasingly shifting to proteomics with theaccomplishment of the gene sequencing of Human Genome Project(HGP).Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. Theinvestigation of functional proteome is of great significance given that functions ofmost of genes are still not well understood. Presently proteomic research focuses ondifferential proteome analysis of human diseases, which is designed to find keyproteins with potentials to be used as markers for diagnosis or targets for medication.Proteomics is also becoming the spotlight of analytical chemistry. The fast development of proteomics relies on breakthroughs for high throughputanalytical technologies. 2D PAGE is the most universal method for proteomics andpossess high resolve power for proteins. Some problems in 2D PAGE, however, arestill difficult to be overcome such as limited pH range, time-consuming and inefficientsensitivity for low abundant proteins. The reality has motivated considerable effort indeveloping new technique to resolve proteomics as alternative approach to 2D PAGE. Liquid chromatography and electrophoresis are powerful technology to resolveproteins. But conventional one dimensional LC or CE are incapable of completelyresolving all the components in a complex mixture due to insufficient peak capacity.The development of two- or multi-dimensional column based separation techniquesgreatly improve the resolve power and peak capacity. During the past decade, a lot ofanalysts 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 part. In the first chapter, advances in 2D and multidimensional separation techniqueswere summarized with details in comprehensive 2D HPLC-HPLC, HPLC-CE andCE-CE system. Capillary array techniques and whole column image detector werealso discussed for high throughput analysis. In the second part of the dissertation, different aspects that can influence theperformance of CIEF were intensively studied and optimized. A primary study on a two-dimensional RPLC/CIEF separation with LIF detectorhas been described in the chapter 3. Capillary RPLC, the first dimension, providedhigh-resolution separations for proteins free of salts. CIEF, the second dimension, hadan orthogonal mechanism to cRPLC and provides excellent resolution capability for -X-<WP=12>复旦大学博士学位论文proteins with several orders concentration. Separation effectiveness of this approachwas demonstrated using complex protein/peptide samples, yeast cytosol and BSAtryptic digest. Apeak capacity of more than ten thousand had been achieved. A laserinduced fluorescence (LIF) detector developed for this system was used to achievehigh sensitive detection. A fmol level of peptide detection limit for BSA digest wasobserved. Two types of dyes, FITC and BODIPY maleimide, to tag the proteins werecompared. The thiol reactive dye was better fit for separations and detections of this2D system. In the chapter 4, based on array capillary isoelectric focusing (ACIEF),comprehensive two-dimensional system of cRPLC as the first dimension and ACIEFas the second dimension was developed for protein mapping. Combining the twoorthogonal high resolution techniques with parallel ACIEF separations, a highthroughput and powerful protein mapping is capable of being achieved. Laser inducedfluorescence (LIF) with whole column imaging detection (WCID) was designed inhouse for detecting protein bands in capillary array without mobilization afterfocusing. This imaging detection could avoid the possible problems likereproducibility, peak distortion, resolution loss arisen by mobilization. Such a systemof CRPLC-ACIEF-LIF-WCID, significantly enhancement of resolution, sensitivity,reproducibility and throughput was obtained. The efficiency o更多还原