液质联用技术在生命磷化学中的分析应用研究

【作者】 刘红霞；

【导师】 赵玉芬；

【作者基本信息】 郑州大学 ， 有机化学， 2005， 博士

【摘要】 生命科学被誉为21世纪的最前沿科学之一。随着人类第一张基因序列草图的完成和发展，生命科学的研究正在进入一个崭新的后基因组学，即蛋白质组学时代。正如基因草图的提前绘制得益于大规模全自动毛细管电泳测序技术一样，借助于现代生物质谱技术，后基因组研究将得到迅猛发展。而现代分离技术和生物质谱的联用技术，极大地扩展了生物质谱的应用领域，正在成为生命科学研究中必不可少的技术支撑。 论文用Atherton-Todd反应，以二乙基亚磷酸酯(DEPH)为磷酰化试剂，在水、醇、四氯化碳和三乙胺存在下，将磷酰基引入含碱性氨基酸的肽段，成功实现了对氨基酸、肽和蛋白质的磷酰化修饰。用电喷雾质谱(ESI／MS)对20种氨基酸进行磷酰化反应的过程进行了在线跟踪，发现只有赖氨酸(Lys)和组氨酸(His)可生成二磷酰化产物。并用ESI／MS和液质联用(HPLC／MS)等现代分析技术，对十种已知序列的肽段和三种常用的蛋白质(胰岛素、溶菌酶和细胞色素C)进行了磷酰化反应产物的分离分析，根据肽段和蛋白质的分子量增加值，确定了肽段和蛋白质中所含Lys、His残基的数目，验证了修饰位点发生在肽链的Lys、His残基上和N端，同时，根据对色谱分离的组分进行MS解析，进一步确认了多种形态的磷酰化产物。研究结果将有助于肽的一级序列分析。 研究建立了N-磷酰化丙氨酸与腺苷反应体系中的单腺苷酸和二腺苷单磷酸等产物的HPLC／ESI／MS分离分析方法，并对2′-AMP，3′-AMP，5′-AMP和3′-5′ApA的MS裂解机理进行了研究。结果表明，HPLC／ESI／MS可以快速、简便、准确地鉴别和定量该复杂反应体系中痕量的2′-AMP、3′-AMP、5′-AMP、3′-5′ApA。首次选择了六种不同极性的N-磷酰化氨基酸与四种核苷混合液进行反应，并用所建立的HPLC／ESI／MS分析方法对混合液反应体系进行了分离分析，通过对可能生成的核苷酸产物XMP、XpY进行离子抽提和MS信息分析，结果不仅观察到了单核苷酸(XMP)和二核苷单磷酸(XpY)的产物生成，而且发现不同反应体系所产生的核苷酸种类和含量都不同，结果表明了不同极性的氨基酸与核苷混合液反应时所表现出的活性差异。该研究结果将为“磷酰化氨基酸为生命起源过程中核酸、蛋白质共同起源”学说，提供一定的实验数据和先进的研究方法。 对地塞米松磷酸钠(DexP)的ESI／MS参数进行了优化，探讨了DexP的质谱裂解规律，研究建立了DexP在不受豚鼠外淋巴液背景干扰情况下的HPLC分离分析条件。利用更多还原

【Abstract】 Life science has been recognized as one of the most frontiers of the 21^st century. With the completion of the mapping of the human genome, life science is progressing into the proteomics age. Similar to the first gene mapping which was completed ahead of schedule using a capillary electrophoresis sequence technique, proteomics research will also develop quickly because of the introduction of the modern bio-mass spectrometry （bio-MS） technique. Together with modern separation techniques, the application scopes of the bio-MS technique will be extended, and high performance liquid chromatography/mass spectrometry （HPLC/MS） coupling technique is becoming a necessary support technique for life science research.In chapter two, in the solution of water, ethanol, carbon tetrachloride and triethylamine, the phosphoryl group was introduced into the peptide segments consisted of basic amino acids using the Atherton-Todd reaction with diethyloxyphosphite （DEPH） as a phosporylation reagent. Phosphorylation modification for amino acids, peptides and proteins were successfully achieved using DEPH. Using electrospray ionization mass spectrometry （ESI/MS）, HPLC/MS and other analytical techniques, the phosphorylation products have been characterized. Of the 20 common amino acids, only lysine （Lys） and histidine （His） could be phosphorylated to produce diphosphorylation products. For the 10 peptides and 3 proteins of known sequence, it was found that the modification sites were on residues of Lys and His on the side-chains. According to the incremental value of the molecular weight, the numbers of the residues of Lys and His in peptide and protein could be determined. These findings were useful to peptide sequence determination and protein structure characterization. It also has potential value for research and development of peptide medicine.In chapter three, a study was conducted to establish a HPLC/ESI/MS methodology for simultaneously determining trace monoadenosine and diadenosine monophosphate in a model reaction between N- （O, O-diisopropyl） phosphoryl alanine（Dipp-Ala） and adenosine（A）. The possible cleavage pathways were depicted and the specific ions were obtained. An optimal and practical HPLC/ESI/MS method was established for separating and determining the positional isomers of 2’-AMP, 3’-AMP, 5’-AMP and 3’-5’ ApA from the complex reaction solution. We first investigated the aqueous reaction between Dipp-aa and the mixed更多还原