【作者】 贾辰熙；

【导师】 何志敏； Lingjun Li；

【作者基本信息】 天津大学 ， 化学工程， 2010， 博士

【摘要】 现代质谱技术具备高灵敏度、高精确度、高通量、高度自动化等诸多优势,在蛋白质组学、神经化学、生理学、核酸化学、细胞信号转导等生命科学领域得到了广泛应用。本文采用综合质谱法及其相关技术,对六类具有复杂结构的生物活性多肽样品:抗癌海洋环肽(第二章)、环肽线性前体(第三章)、二硫键神经肽(第四章)、大型神经肽(第五章)、合成消旋肽(第六章)、酶解肽(第七章)进行解析研究,旨在开发基于综合质谱法的新颖肽谱分析方法学,并深入研究与质谱学密切相关的多肽质谱碎裂机理,同时在各章中穿插介绍了环肽的合成、交联酶聚体的制备、反相色谱保留机理、神经肽生理学测试等辅助性研究工作。综合质谱法有效地整合、运用多种样品前处理、样品分离等手段,质谱电离源、离子分离器、质量分析器等技术,数据分析、肽测序软件、数据库搜索等工具,气相离子化学、酶学、MALDI基质化学、MALDI成像学等学科;并将这些相关技术、手段、工具、理论应用于复杂多肽样品的表征及其生理学活性的研究。本研究所建立的方法学与理论体系以及具体研究内容与结论如下:第一,采用多级质谱法对具有抗癌活性的海洋环肽进行序列测定,依据bx→bx-1断裂路径,每一级MS由b离子的C端碰撞掉一个氨基酸残基,得到2套b离子序列信息,由此测得环肽序列;利用半经验量子化学计算了断裂与重排的路径和机理,并尝试提出过渡结构假设;研究了环肽CAD中选择性开环的现象,提出Asn诱导开环的碎裂路径。第二,针对多级质谱测序中观察到的b离子丢失中部氨基酸残基的现象,提出b离子环化碎裂路径,并设计多项实验加以验证,包括肽链N端乙酰化、多级CAD、设计重排b离子、调节CAD活化时间、CAD碎裂合成环肽;观察到由ESI产生的质子化环肽的碎裂模式与b离子环化产物的碎裂模式一致,有力证明了所提出的b离子环肽中间体以及环化路径假设的成立;尝试设计实验探索此环化路径的规律性,并提炼出反应开环过程规律性的“Pro和Asn/Gln效应”;此外,观察到a型离子丢失中部残基的现象,提出a离子环化碎裂路径;此a离子环肽中间体为固定电荷结构,故不可以被“Pro效应”影响,导致易于在肽键Pro-Xxx而不是Xxx-Pro开环。第三,建立还原基质法对含二硫键多肽进行扫描,结合表达序列数据库对肽序列预测,在蓝蟹围心器提取液中发现并解析了新颖的咽侧体抑制素CbAST-C1;电生理学实验结果证明此咽侧体抑制素肽都可以阶段性地抑制蓝蟹口胃神经节的幽门节律;应用肽浓度为10-6 M,当起始幽门节律大于0.8 Hz时,两个AST肽对幽门节律的影响很小;但当起始幽门节律小于0.7 Hz时,两肽均可完全抑制幽门节律。第四,建立多面质谱法对蓝蟹神经组织中CHH家族肽进行表征与解析;从窦腺中鉴定出了SG-CHH、SG-MIH、SG-CPRP、SG-tCPRP和SG-PPRP;从围心器中鉴定出了PO-CHH、PO-CPRP和PO-tCPRP;建立酶解后甲醛同位素标记法对SG中的CHH家族肽和相关大型肽进行定量分析,并建立质谱成像法对PO中CHH家族肽和相关大型肽进行分布分析,为今后生理学活性研究奠定了方法学基础。第五,针对弱疏水性合成七肽,提出“早期吸附”过程,以此优化色谱条件得到4个峰形良好的色谱峰;质谱分析表明:4谱峰具有相同的m/z 761.5 [M+H]+值,对每一消旋产物进行在线多肽测序;利用质谱、色谱相结合的方法对固相合成关键性问题进行研究,包括水杨醛法测定树脂取代率,合理搭配缩合体系提升酯化缩合效率至99%,合理选择氨基酸侧链保护剂,减少合成的预活化时间为5分钟以避免了消旋化。第六,基于胰蛋白酶CLEA技术的制备工艺、性质及结构形貌的系统研究,建立CLEA板上酶解法用于酶解肽谱的解析,考察了CLEA抗自水解性能、高温酶解稳定性以及CLEA颗粒对MALDI电离的影响程度;使用7种蛋白样品测试所建立的CLEA-trypsin板上酶解法,可得到同量级的多肽指纹图谱解析结果:46%的平均序列覆盖率,14个平均酶解肽段数目;与传统的14小时酶解相比,该法显著提高了分析通量(5 min),减少了样品消耗量(下至4 pmol)。更多还原

【Abstract】 Modern mass spectrometry (MS) technology has several distinct advantages, including high sensitivity, accuracy, through-put, automation, etc, which has been applied to most of life science research fields, such as proteomics, neurochemistry, physiology, nucleic acidic chemistry, cell signaling, etc. This work focuses on establishment of comprehensive mass spectrometry and related technologies to analyze six peptide families with complicated structures, involving antitumor marine cyclic peptides, linear precursor of cyclic peptides, disulfide-linked peptides, large neuropeptides, racemized peptides, proteolytic peptides, and aims to develop novel MS-based methodology for peptide mapping. In addition, the peptide fragmentation mechanism in MS was also studied in deep, and several related research works were introduced including synthesis of cyclic peptides, preparation of cross-linked enzyme aggregates, retention mechanism of reversed-phase chromatography, physiology test of neuropeptides, etc.Comprehensive MS is a useful tool box that contains protocols of sample pretreatment and separation, techniques of ionization source, ion selection, and ion analyzer, strategies of data analysis, peptide sequencing programming, and database searching, theories of gas-phase ion chemistry, enzymology, matrix chemistry, MS imaging, which was combined and further used to characterization of peptides and biological functions. The research details are described as following,First, the multistage MS method was established for sequencing of antitumor marine cyclic peptides. Based on bx→bx-1 fragmentation pathway, the b ion can dissociate to lose one C-terminal amino acid residue in every stage of CAD, producing two sets of b ions. The cyclic peptide sequence can be determined by observation of these b ions. The semiempirical calculation was used to investigate the normal fragmentation and abnormal rearrangement pathways. The selective ring opening of cyclic peptides was studied, and Asn-induced ring opening mechanism was proposed.Second, during characterization of some peptides in an ion trap, it was noted that many internal amino-acid residues could be lost from singly charged b ions. The unique fragmentation consisting of multiple steps is induced by a cyclization reaction of b ions, the mechanism of which has been probed by experiments of N-acetylation, MSn, rearranged-ion design and activation-time adjustment. The fragmentation of synthetic cyclic peptides demonstrates that a cyclic peptide intermediate (CPI) formed by b ion cyclization exhibits the same fragmentation pattern as a protonated cyclic peptide. Although no rules for the cyclization reaction were discerned in the experiments of peptide modification, the fragmentations of a number of b ions indicate that the“Pro and Asn/Gln effects”can influence ring openings of CPIs. In addition, large-scale losses of internal residues from different positions of a-type ions have been observed. The fragmentation is initiated by a cyclization reaction forming an a-type ion CPI. This CPI with a fixed-charge structure cannot be influenced by the“Pro effect”, causing a selective ring opening at the amide bond Pro-Xxx rather than Xxx-Pro.Third, using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), present in the pericardial organs of the crab, Cancer borealis. Electrophysiological experiments demonstrated that the AST peptides inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10-6M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies < 0.7Hz.Forth, the multifaceted MS method was established for characterization of CHH-family peptides in nervous system of blue crab. The peptides SG-CHH, SG-MIH, SG-CPRP, SG-tCPRP and SG-PPRP were identified in pericardial organ, and the peptides PO-CHH, PO-CPR and PO-tCPRP were identified in sinus gland.The isotopic labeling strategy and MS imaging strategy were established for quantitation and distribution analysis of CHH-family peptides and homologous large peptides, which laid the foundation for future study on physiological function. Fifth, the retention characteristic of hydrophilic heptapeptide on RP-HPLC was discussed, and the process of‘pre-adsorption’was proposed in order to take full advantage of contributions of both retention mechanisms of partition and adsorption to separation. Thus by this method four ideal RP-HPLC peaks were detected. Meanwhile, the MS information indicated that the four peaks had the same value of m/z 761.5[M + H]+. Then through simultaneous peptide sequencing of each racemization products and figure superposition of tandem MS (MSn), the two sets of segment ions, b and y, were observed except b1 and y1, which showed that the four racemization products of synthetic heptapeptide were successfully identified.Sixth, immobilized enzymes have been utilized in proteomics due to several distinct advantages. However, most immobilization methods require an inactive medium to support the enzyme, which occupies 90–99% enzymatic volume, lowering effective enzyme concentration. Herein, we introduce a carrier-free enzyme immobilization technology into proteomics for rapid proteolysis. Cross-linked trypsin aggregates (CLEA-trypsin) were prepared by a simple and low-cost method, showing excellent proteolysis efficiency, improved thermal stability, and reduced enzyme autolysis. A high throughput proteomic strategy was developed by incorporating on-plate CLEA-tryptic digestion and high-accurate MALDI-FTMS peptide mass fingerprinting, and an average sequence coverage of 46% was obtained by digestion of seven protein standards for 5 min at 77°C.更多还原