【作者】 张国安；

【导师】 杨芃原；

【作者基本信息】 复旦大学 ， 分析化学， 2003， 博士

【摘要】 本博士论文工作的主要贡献为发展了蛋白质凝胶内酶切方法以及胶内酶切产物的在线预浓缩技术；另外对新型介孔无机材料在MALDI中的应用也进行了研究；利用基于双向电泳和生物质谱的方法策略对缺血心肌线粒体以及高、低转移潜能肝癌组织进行了差异蛋白质组分析。后基因组学的主要流派蛋白质组学目前显得相当活跃。蛋白质组学分离和分析细胞与组织的全部蛋白并直接找到一组或几组功能蛋白，并研究它们与功能基因(组)的内在联系 。在目前功能基因尚不很清楚的情况下，直接发现功能蛋白组具有重要的意义。目前，蛋白质组学的研究主要集中在通过对蛋白质性质、丰度的考察来揭示它的功能，进而找到与人类重大疾病相关的差异蛋白，使之成为药物筛选的靶分子，指导基因组的分析，而不是仅仅尾随DNA序列分析的结果。蛋白质组学也正在成为分析化学研究领域的最前沿研究而蛋白质组学的科学研究之所以能够取得蓬勃的发展，主要依赖于高通量分离和分析技术的突破性进步。首先是双向凝胶电泳技术的完善，使得蛋白质的大范围、高通量分析成为可能。其次是质谱技术尤其是软电离技术的发展，使之成为检测蛋白质分子的重要手段。最后是生物信息学的建立使得国际性的科学大协作得以形成。目前，世界各主要国家都不惜巨资进行蛋白质组的研究。蛋白质组学研究的进一步深入，将对了解疾病的发生和药物的筛选起到决定性的作用。本论文工作包括两个部分：第一部分为蛋白质组学技术平台的建立与发展，建立了基于双向电泳和质谱的蛋白质组学技术平台，对胶内酶解方法以及其浓缩方法进行了改进和发展，并对无机MALDI基质作了初步研究。第二部分为疾病比较蛋白质组初探，利用基于双向电泳和生物质谱的方法策略对缺血心肌线粒体以及高、低转移潜能肝癌组织进行了比较蛋白质组分析。第一部分 蛋白质组学技术平台的建立与发展聚丙烯酰胺凝胶电泳，包括一维和二维，是目前蛋白质研究中用来分离复杂蛋白体系的最重要的手段。而对胶上蛋白进行胶内酶解以及质谱分析，也已经成为复杂体系中蛋白鉴定的最常用的方法。由于考马斯亮蓝的存在会影响酶解反应，所有现有的胶内酶解方法都要求在酶解反应之前彻底除去凝胶中的考马斯亮蓝。这个过程增加了工作量，耗时而且会造成蛋白丢失，影响蛋白鉴定的灵敏度。本工作提出了一种简化的胶内酶解方法，在酶解前不需除去考马斯亮蓝。利用简<WP=7>化方法对500 ng BSA 进行酶解，并用HPLC/ESIMS进行分析。结果表明使用简化方法时酶解仍然可以有效进行。另外，考马斯亮蓝在反相HPLC中有强保留，不会与肽段同时洗脱，所以不会对HPLC-ESIMS分析造成干扰。利用一系列不同上样量的马心肌红蛋白对简化方法与传统方法进行了比较，发现简化方法倾向于产生更多的胰蛋白酶漏切位点，并可以因此提高序列覆盖率。对简化方法在MALDI-TOFMS上的适用性进行了验证，在用ZipTip脱盐时使用33%的乙腈洗脱以防止考马斯亮蓝与肽段同时洗脱。50 ng BSA 可以得到令人满意的结果，显示了简化的方法可方便地用于MALDI-TOFMS检测，并拥有足够的灵敏度。用该方法对大鼠心肌线粒体蛋白双向电泳图上的两个蛋白点进行了成功鉴定，表明简化方法完全可以适用于实际生物样品的分析。在胶内酶解产物的质谱鉴定前，通常需对样品进行浓缩。常规的浓缩方法费时费力，而且会由于容器表面吸附造成大量样品损失。本论文发展了一种基于强阳离子交换色谱(SCX)的凝胶内酶切产物的在线预浓缩方法，可以方便地应用于蛋白的LC-ESIMS分析。由于胶内酶解肽段的提取液（5% FA或TFA/50%乙腈）恰好与SCX肽段吸附的条件相吻合，因此肽段提取液可以不经过任何处理直接用SCX 柱浓缩，然后用1 M 醋酸铵溶液将吸附的肽段洗脱进入反相LC-MS系统进行分析。独特设计的预浓缩/分析体系利用两个六通阀将SCX和反相色谱相连，可以很方便地实现30倍的在线预浓缩。利用100 ng 的胶内牛血清蛋白对该方法进行了验证，与传统的浓缩方法相比，该方法可以获得9个肽段，而传统的冷冻抽干方法仅得到5个。其原因有两点：（1）新方法可以大大减少传统方法的样品干燥和转移过程中由于容器表面吸附引起的损失。（2）在SCX浓缩过程中，可以除去样品中的非离子性杂质，有利于改善样品峰的信噪比。另外，该方法具有省时、装置简单、易于自动化和有利于反相色谱柱的保护等优点。有机基质的出现大大推动了基质辅助激光解析电离质谱（MALDI-MS）在生物大分子分析中的应用。然而有机基质自身电离会在质谱的低质量范围产生背景，影响MALDI-MS对小分子的分析。虽然有人研究了利用无机材料作为MALDI基质的可能性，但其灵敏度非常低。本论文研究了一系列无机介孔材料（TiO2, CeTiO, ZrTiO, TiSiO, ZrO2, AlTiO, AlSiO和SiO2）作为MALDI基质的可能性。样品为PPG（分子量范围500 至3000 Da）和一个标准肽（分子量980 Da）。被测材料对分析物的电离能力表现出极大差异，TiO2 和CeTiO 灵敏度最高，ZrTiO, TiSiO, ZrO2, AlTiO 和AlSiO 的电离能力低得多，而SiO2无法使样品电离。其该结果提示材料的灵敏度与UV吸收正相关。被分析物通常以钾离子加合物的形式电离，偶见钠离子加合更多还原

【Abstract】 The main contributions of this dissertation are: a modified in-gel digestion protocol was developed; a new online preconcentration approach was developed; a series of new mesoporus materials were investigated for their potential as MALDI matrices; and differential proteomic analysis was carried out for ischemic heart mitochondrion and liver carcinomas with high and low potential for metastasis. Scientific interest has been increasingly shifting to proteomics with fast measurement of genomic sequences. 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 meditation. Proteomics is also becoming the spotlight of analytical chemistry. The fast development of proteomics relies on breakthroughs for high throughput analytical technologies which include two-dimensional electrophoresis, biological mass spectrometry and bioinformatics. This dissertation consists of two parts. In the first part, a proteomic platform based on two-dimensional electrophoresis and mass spectrometry was established. With this platform, in-gel digestion methodology was investigated and a modified in-gel digestion protocol and a new online preconcentration approach were developed. Also a preliminary investigation was carried out for inorganic MALDI matrix. In the second part, differential proteomic analysis was carried out for ischemic heart mitochondrion and liver carcinomas with high and low potential for metastasis.Part I: Establishment and development of proteomic platform.Polyacrylamide gel electrophoresis, either in one or two dimensions, is the most commonly used technique for separation of complex protein samples. And in-gel digestion combined with mass spectrometry has become a major route for protein identification. Various versions of protocols for tryptic in-gel digestion of CBB-stained proteins have been documented and all of them require a thorough removal of the dye with extensive washing steps prior to proteolysis. The washing<WP=11>steps slow down the procedure and cause sample loss. Accordingly a simplified protocol was proposed which does not demand a removal of the dye. The simplified protocol was tested using 500 ng BSA. The result of HPLC/ESIMS showed tryptic digestion was hardly affected by CBB. Because CBB is strongly retained in RPLC, it does not affect the analysis of peptides in MS. A comparison between the simplified protocol and the conventional one revealed that the simplified protocol tended to produce more incompletely cleaved peptides, which is favorable for improvement of sequence coverage. Experiments with 50 ng BSA showed that the simplified protocol can be used for MALDI-TOFMS with sufficient sensitivity. The simplified protocol was further applied to the analysis of two protein spots on a 2DE map of rat heart mitochondrion.A new method was developed for on-line pre-concentration of peptides from protein digested in-gel prior to HPLC-ESI-MS-MS analysis using strong cation exchange (SCX) chromatography. Peptides were directed loaded onto a capillary SCX column without any pretreatment, and then displaced using 1 M ammonia acetate to a reversed phase LC-MS system. A 30 fold concentration was achieved accordingly. The method was validated with tryptic peptides of bovine serum albumin (BSA), in which 100 ng of BSA in polyacrylamide gel was successfully identified. Compared with the conventional technique, more peptides were detected with less sample loss and the ability of removing contaminants by SCX chromatography. Consequently, the new approach offers less time consuming, easier to automate, and more favorable for LC column protection and analysis reproducibility.The introduction of organic matrices to MALDI-MS greatly advanced bimolecul更多还原