【作者】 于雁灵；

【导师】 杨芃原；

【作者基本信息】 复旦大学 ， 分析化学， 2004， 博士

【摘要】 蛋白质组学技术是生命科学中的分析化学的主要内容,是当前的研究前沿,是化学生物学近年来的新研究方向。本论文工作是在建立蛋白质组研究实验室的过程中,建立和发展了蛋白质组、比较蛋白质组和定量蛋白质组研究的相关技术平台,并将其应用于人类重大疾病的研究。定量蛋白质组学是通过某种方法或技术,对生物样品(细胞、组织或体液等)在某些过程中蛋白质的含量进行比较分析。从蛋白质组水平上对基因表达进行准确的定量分析,是比较蛋白质组学的重要内容,是当前研究重大疾病致病机制以及药理控制机制的必要手段及研究前沿。本论文工作的主要贡献是,建立了一套稳定可靠的蛋白质顺序提取-双向凝胶电泳分离-半自动胶上酶解-质谱鉴定的高通量技术分析平台;建立了一种新的、基于肽段乙酰基化的稳定同位素标记定量蛋白质组分析技术,并在实际生物样品体系中进行了验证;将建立、优化的蛋白质组定量分析技术应用于动脉粥样硬化、冠心病以及肝癌的复发转移相关的多个实际生物样品的比较分析,并从分子水平上评价了几种药物的药效。本文还研究了对基于生物素-抗生物素蛋白特异性结合的亲和色谱分离体系,结果显示该体系有望实现与乙酰基化同位素标记技术的有效对接,建立新的定量分析技术。一、在优化和发展传统双向凝胶电泳蛋白质组、比较蛋白质组方法的基础上,建立了蛋白质组顺序提取-双向凝胶电泳分离-半自动胶上酶解-质谱鉴定的高通量技术分析平台,为蛋白质组学方法学研究和应用研究奠定了实验基础鉴于蛋白质组的高度复杂性,在进行双向凝胶电泳前对蛋白质进行预分组是十分必要的。顺序提取按蛋白质溶解能力的不同将复杂的蛋白质组预分组,是目前常用的比较有效的样品预分组的方法之一。如何根据实验需求合理选择不同溶解能力的顺序提取液,不同的蛋白质提取液可否直接进行双向凝胶电泳分离并得到良好的分离效果等问题,是影响蛋白质组分析的重要因素。本论文提出了一套完整的蛋白质三步提取方案,可以确保几乎所有的蛋白质进入相应的溶液中。其中,T1体系 (1 mmol/L PMSF的水溶液) 由于不含任何的蛋白质去垢剂、变性剂等,背景溶液组成简单,可以很方便地采取各种色谱分离的手段进行分离,也可以在直接补加一定量的蛋白质去垢剂、变性剂后采用双向凝胶电泳进行分离;T2 (7 mol/L 尿素,2 mol/L硫脲,2% CHAPS,2% SB3-10,<WP=7>50 mmol/L DTT,1 mmol/L PMSF) 背景溶液的组成与双向凝胶电泳分离的缓冲体系相容性较好,因此可以利用双向凝胶电泳优越的分离能力和直观的检测方式直接对E2进行分离分析。由于高丰度蛋白质多存在于胞浆中,可溶性较好,E2中大量高丰度蛋白质已被提取,间接提高了上样量,使一些中、低丰度的蛋白质得到检测,提高了蛋白质的检出率;在T2提取液的组成中加入离子性去垢剂SDS作为第三级提取液T3,将T2提取后的不溶物基本上全部溶解,同时由于SDS的存在,稀释不会导致蛋白质的析出,通过稀释,可以直接采取多维色谱进行分离分析。作为该平台中联系双向凝胶电泳分离和质谱鉴定的桥梁,胶内酶解过程是影响肽段回收率的主要因素。本论文将原手工胶上酶解过程经适当调整,应用于高通量半自动酶解分析,提高了分析速度和通量;实验考察了对半自动胶上酶解过程中严重影响肽段回收率和蛋白质鉴定的确定性的诸多环节,包括酶液使用量的多少、酶解时间的长短、是否去盐等问题,优化了实验参数。即当用96孔板酶解一粒直径1.4 mm的胶粒时,使用3 ?l的胰蛋白酶液(12.5 ng/?l)足以使其完全酶解,酶解时间长(酶解过夜)有助于得到较多的肽段,胶上酶解所得的肽段是否去盐对质谱分析所得的谱图质量的影响,主要取决于肽段溶液中含盐量及溶液中肽段的相对多少等等。二、双向凝胶电泳-质谱鉴定定量蛋白质组学在疾病研究中的应用 本论文应用双向凝胶电泳-质谱鉴定比较蛋白质组的方法对心血管、肝癌等两类重大疾病相关的细胞系或组织的蛋白质组进行了比较分析,奠定了致病过程中关键蛋白质筛选的基础,结果对药物的研发、药效的评价也具有指导意义。1、动脉粥样硬化(atherosclerosis,AS)泡沫细胞模型比较蛋白质组研究。AS是心脑血管疾病的共同病理基础,而泡沫细胞是AS斑块内出现的特征性病理细胞。AS早期,单核细胞首先粘附于血管内皮,然后迁移至内皮下分化为巨噬细胞,后者主要通过清道夫受体无节制地吞噬大量氧化低密度脂蛋白(ox-LDL),形成巨噬源性泡沫细胞,进而堆积成AS斑块。利用双向凝胶电泳对正常巨噬细胞、泡沫细胞进行了差异蛋白质组分析。对其中26个高表达蛋白质点及11个差异蛋白质点进行手工胶上酶解,1D LC-ESI/MS鉴定;对鉴定出的差异蛋白质按功能进行了归类,并讨论了其在泡沫细胞形成过程中可能的作用。研究了具有抗AS功效的银杏叶提取物(GbE)、香豆素提取单体(IMP)作用后泡沫细胞蛋白质组的变化情况。通过比较分析双向凝胶电泳分离所得的图谱,发现两种药物对差异蛋白质皆有一定的回调作用,但<WP=8>回调的蛋白质种类和程度却不完全相同,提示两种药物的抗AS作用在机制上存在差异。本工作用双向凝胶电泳分离技术成功地筛选并确定了不同药物的作用靶点。2、冠心病(Coronary Heart Disease, CHD)家兔主动脉血管、肝脏组更多还原

【Abstract】 Proteomic techniques investigation is the main content of analytical chemistry in the field of Life Sciences, and becomes the leading area in recent years. This dissertation is focused on the investigation of proteomics and quantitative proteomics methodology together with its applications to the human serious diseases in the course of Proteomics Lab foundation.The goal of quantitative proteomics is to examine all of the changes induced by a certain perturbation in a biological system. Quantitative proteomics is the main aspects of comparative proteomics, and can provide major approaches to the elucidation of disease mechanisms and to the identification of new diagnostic markers and therapeutic targets. The main contributions are, Establishment of a technique platform with high throughput involving protein sequential extraction - two dimensional gel electrophoresis (2DE) separation - semi automatic in gel digestion - MS identification; Establishment of a new quantitative proteomic technique based on the acetylation stable-isotope labeling combining with 1D PAGE separation, which has been successfully confirmed by applying it to real bio-samples; Comparative proteomic investigations of several real-bio-samples involving Atherosclerosis, Coronary Heart Disease and metastasis of liver cancer, and evaluation of some kinds of pharmaceutics from the protein level. Meanwhile, investigations on affinity isolation based on the firm combination between biotin and avidin, in order to eventually realize the efficient combination of affinity isolation and acetylation isotope labeling. The main contents can be descried as follows:1. Establishment of a high throughput technique platform including protein sequential extraction -2DE separation - semi automatic in gel digestion - MS identification,found a solid experimental basis for proteomics investigations <WP=13>(1) Sample pre-fractionation is essential to the proteomic investigation because of the complexity of a certain proteome, and sequential extraction holds a great promise on protein fractionation prior to further analysis. Reasonable selection of the extraction solution is the key factor to the successive analysis of proteomics study.A feasible three-step protein extraction protocol was proposed. Almost all the proteins were entered into the related extracts. Briefly, an aqueous solution containing 1 mmol/L PMSF was used as the first extract solution (T1) and only restricted sorts of proteins were extracted because of no denaturant and detergent was contained, so that E1 was suited for the direct separation by chromatography or electrophoresis, etc. because of its clean background. Also, in case a definite quantity of urea and CHAPS were added into the rehydration system of E1, 2DE separation on E1 can be carried out. The mixture of 7 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 2% SB3-10, 50 mmol/L DTT, 1 mmol/L PMSF was used as the second extract solution (T2) which possesses the greatest solubility and can still be used to the 2D gel separation. Moreover, the proteins with relatively low abundance can thus be detected because of the removal of large quantity of cytosolic proteins. At last, 2% SDS substituted 2% SB3-10 of T2 was used as T3, and nearly no residue was remained. E3 obtained can be analyzed via either 1D PAGE-LC/MS or 2D LC/MS. Thus, almost all proteins in a certain proteome can be solubilized by using such a three-step sequential extraction protocol described above.As the linkage of 2DE separation and MS identification, in-gel digestion remains the primary procedure for peptide coverage. The original protocol of manual in-gel digestion has been re-adjusted to some extent, and was applied to the semi-automatic analysis, resulting in high speed and throughput; Many factors during the semi-automatic in-gel digestion, such as trypsin quantity, digestion duration, pre-desalting or not prior to MS, et al, which would interfere with the peptide coverage and definition of MS identification severely are tested.2. Applications of comparative prote更多还原