【作者】 林建峰；

【导师】 陈清西； 赵辅昆；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 基于凝胶蛋白质组学技术提供了蛋白质的定量和定性信息,在蛋白质组学研究中,尤其是在临床穿刺样品的分析中起着核心作用。本文针对基于凝胶蛋白质组学中技术性强、手工依赖性大、对分析质量产生重要影响的3个环节,即双向电泳(2DE)、蛋白质显色及蛋白质胶内酶解,进行方法学研究;同时应用基于凝胶蛋白质组学技术对前列腺穿刺样品(PNBX)进行前列腺癌的蛋白质组学研究。不仅建立了适用于不同生物样品的高重复性、高分辩率的基于凝胶蛋白质组学分析平台,而且首次揭示了蕴含在PNBX中的蛋白质组信息,发现了一些潜在的前列腺癌分子标记物。在方法学研究部分,本文首先建立并且比较分析两种双向电泳体系ISO-DALT和IPG-DALT。结果表明IPG-DALT具有更高的分辨率和重复性,但是,碱性条纹是IPG-DALT中的一个顽疾。围绕碱性条纹产生的原因,本文进行了一系列的优化,包括提高DTT的使用量、缩短等电聚焦时间、用还原剂TCEP代替DTT并用4-VP烷基化蛋白质以及碱性端杯上样,从而建立了高重复性、高分辨率、适用于体液(血清和尿液)、细胞及组织蛋白质样品的双向电泳分离方法。其次,为了更好地衔接2DE和质谱分析,本文以BSA和293T细胞蛋白质样品作为生物模型,比较分析了目前被广泛使用的7种可见蛋白质显色方法(Neuhoff CCB、Blue silver、LKB SN、He SN、Yan SN、Vorum SN及Blum SN)的灵敏度、背景、线性关系以及MALDI-TOF质谱兼容性。从而对各种显色方法的染色效率及质谱兼容性有了全面的了解,对蛋白质显色的化学原理有了较为深刻的认识。其中Yan SN染色效率高、质谱兼容性良好;Neuhoff CCB质谱兼容性好且与Yan SN匹配性好。这两种显色方法被分别选用于后续前列腺穿刺样品蛋白质组研究的分析胶和半制备胶的蛋白质显色。最后,为了提高蛋白质质谱鉴定的成功率和准确性,本文在质谱样品制备程序上进行摸索,包括探究脱色与否的质谱兼容性,分析还原和烷基化处理对蛋白质质谱鉴定的影响,及检测不同酶解时间的酶解效果,从而建立适用于基于凝胶蛋白质组学研究的蛋白质高效胶内胰酶酶解程序。前列腺癌(PCa)是世界范围的男性高发疾病,生物学特性复杂,是肿瘤研究的热点之一。PNBX是PCa诊断阶段获得的临床样品,其蕴涵的蛋白质信息,对我们了解PCa极为重要。运用所建立的基于凝胶蛋白质组学分析平台,本文选萈NBX样品,进行PCa的蛋白质组学研究。首先,本文收集26支来自PCa和前列腺增生(BPH)病人的PNBX样品。其次,因为前列腺穿刺存在约30%的假阴性,本文通过免疫印迹测定样品中肿瘤标记物AMACR的表达情况,筛选用于蛋白质组学分析的PNBX样品。继而,对9支PCa PNBX样品和14支BPH PNBX样品进行差异蛋白质组学分析。2DE结果显示52个蛋白质点在PCa与BPH之间具有显著差异。包括这些点在内的总共228个蛋白质点被进一步用MALDI-TOF/TOF质谱分析鉴定。其中最值得关注的两组蛋白质是潜在的雄激素受体调控蛋白[FLNA(7-15)和FKBP4]和参与线粒体脂肪酸β氧化的酶(DCI和ECHS1)。此外,FLNA(7-15)、FKBP4、ECHS1、DCI、CCT6A、和MFAP4在所有PNBX样品中表现出一致的表达趋势,具有一定的作为诊断标记物的应用价值。实验还观察到:在PCa中抗氧蛋白PRDX各亚型呈现出不均衡表达;PCa中存在不同修饰状态下的HSP27及HSP70.1,而且各种修饰状态呈现不同的表达模式;一些之前被认为是PCa分子标记物的蛋白质如PSA和ACPP在PCa与BPH的PNBX间没有显著差异。通过免疫印迹实验,本文进一步验证了FLNA(7-15)、FKBP4及PRDX4的差异表达。本部分的结果表明:基于凝胶蛋白质组学方法适用于PNBX样品的蛋白质组分析;通过该方法发现的蛋白质具有潜在的作为前列腺癌诊断、预后标记物或治疗靶点的意义。更多还原

【Abstract】 Gel-based proteomics generates qualitative and quantitative protein behavioral data, and as such it plays a central role in proteomic studies, particularly in the analysis of clinical biopsy specimens. In this study, efforts focused on 2DE, protein visualization, and protein in-gel digestion were made to ensure success of gel-based proteomics. With the optimized techniques, I conducted the proteomic analysis of prostate cancer using prostate needle biopsy (PNBX) specimens. This study not only establishd reliable gel-based proteomic approaches for various biological samples, but also defined the proteomic features implicated in PNBX specimens, and profiled the candidate biomarkers of prostate cancer.In the part of methodology development, first, comparisons referring to resolution and reproduction were made between two 2DE techniques, ISO-DALT and IPG-DALT, and the later was selected for its better results. To address the horizontal streaks in the alkaline region, four strategies were introduced and compared including elevating the concentration of DTT, shortening IEF duration, using the reducing agent TCEP and alkylating agent 4-VP in sample preparation, and anodic cup-loading. Thereafter an IPG-DALT separation system suiting to analyze the protein composition of biofluids (serum and urine), cells, or tissue was established. Second, using BSA and 293T cell proteins as biological models, I compared seven widely used protein visualization methods: Neuhoff CCB, Blue silver and five popular silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN) with respect to sensitivity, background, dynamic range, and MALDI-TOF MS compatibility. With the rusults, I not only obtained the full-scale information about the stain efficiency and MS compatibility of these seven methods, but also got insight into the mechanism of the protein staining. Yan SN was selected in analytical gels for its good stain efficiency and acceptable MALDI-TOF MS compatibility, and Neuhoff CCB was selected in semi-preparative gels for its excellent MS compatibility and good conformability with Yan SN. Last, to improve the MS identification, studies were performed to determine the compatibility of in-gel trypsin digestion of stained protein spots with MS, to analyze the effects of reduction and alkylation on 2DE spot identification, and to evaluate time necessary for in-gel trypsin digestion for protein characterization with MS.Furthermore, the optimized gel-based proteomic approaches were used to analyze the PNBX specimens. Prostate cancer (PCa) is a significant cause of the morbidity and mortality worldwide. PNBX is a specficial clinical material obtained in the diagnose stage. Proteomic analysis of PNBX specimens is critical to our understanding of human prostate cancer. Twenty-six PNBX specimens from patients with PCa or benign prostatic hyperplasia (BPH) were collected. It is well-known that PNBX have false-negative rates approaching 30%, depending on the technique used. So protein samples were then subjected to immunoblot analysis for the malignant marker, AMACR, to test the tissue homogeneity. 14 BPH specimens and 9 AMACR-positive PCa specimens were further subjected to comparative proteomic analysis. 2DE revealed that 52 protein spots exhibited statistically significantly changes among PCa and BPH groups. Total 228 spots were analyzed further by MALDI-TOF/TOF MS. The two most notable groups of proteins identified included latent androgen receptor co-regulators [FLNA(7-15) and FKBP4] and enzymes involved in mitochondrial fatty acidβ-oxidation (ECHS1 and DCI). FLNA(7-15), FKBP4, ECHS1, DCI, CCT6A, and MFAP4 exhibited consistent expression patterns and may be useful for the development of diagnostic markers. An imbalance in the expression of peroxiredoxin subtypes was noted in PCa specimens. Different post-translationally modified isoforms of HSP27 and HSP70.1 were identified, and certain modifications to them were specifically linded to PCa. Furthermore, PSA and ACPP, which were previous defined as PCa markers, lacked significant change between PCa and BPH PNBX specimens. Importantly, changes in FLNA(7-15), FKBP4, and PRDX4 expression were confirmed by immunoblot analysis. The results suggest that the gel-based proteomic approach is useful for developing a more complete picture of the protein profile of PNBX specimen. The proteins identified by this approach may be useful molecular targets for PCa diagnostics and therapeutics.更多还原