多层键合磷酸锆开管毛细管柱制备及在磷酸肽富集分析中的应用

发布时间：2018-05-21 21:45

本文选题：多层开管毛细管柱 + 磷酸锆　；参考：《安徽医科大学》2010年硕士论文

【摘要】： 可逆的磷酸化过程在细胞增殖、分化、代谢、细胞间通讯以及信号转导通路等重要的生物学过程中发挥着重要的调节作用,真核细胞中,有近25-30%的蛋白在其生活史中会经历磷酸化过程。因此,蛋白质的磷酸化修饰是翻译后修饰蛋白质组研究中的热点之一。目前,包括MALDI源和ESI源在内的质谱仪以其高灵敏度、准确度和分辨率而被广泛用于蛋白质组学研究中。由于磷酸化蛋白在生物体内含量很低,且在质谱分析时易受到非磷酸肽的干扰,因而磷酸肽和磷酸化蛋白质的富集成为磷酸化肽段和磷酸化蛋白质分析的一个关键步骤。目前已经有很多富集磷酸肽的方法和技术,但这些方法在应用时仍然存在不少问题,因而需要发展新的富集方法,以满足大规模磷酸化蛋白质组分析的需要。基于此,本文开展了一种磷酸肽富集的新方法研究,并将其应用于复杂样品的分析。本课题发展的方法为一种利用键合磷酸锆开管毛细管柱的制备及用于快速富集磷酸肽的新方法。其过程是通过酸碱活化熔融石英毛细管内表面,使其带有自由的硅羟基,在硅羟基表面引入3-氨丙基三乙氧基硅烷,之后再分别引入甲基丙烯酸甲酯和乙二胺,如此进行三轮反应后,在形成的树枝状大分子表面进行磷酸基团修饰并与锆离子生成磷酸盐,最终合成具有特异性吸附磷酸肽分子的多层开管毛细管柱。进一步对该多层开管毛细管柱的富集条件进行了优化,确定出当开管毛细管柱长度为50 cm时,标准磷酸肽的进样流速在0.3μL/min -0.5μL/min时,均有很好的富集效果。并且在同样的流速条件下,使用不同内径的多层开管柱进行富集时,富集效果没有显著性差异。同时还考察了强阳离子交换色谱分离中分离效果较好的盐,如氯化铵对富集效果的影响,以便该方法与复杂样本的预分离过程相兼容。之后,还用合成的磷酸肽段(FLpTEYVATR)与外标法相结合,对开管毛细管柱的负载量和回收率等进行考察,确定出负载量为48 pmol,回收率为94.1%(50 cm×50μm)。利用α酪蛋白与BSA混合酶切溶液,考察了毛细管开管柱的选择性和检测灵敏度,结果表明此方法对磷酸肽有较高的选择性,且检测限达到10-9M。在优化的实验条件下,利用牛α酪蛋白对开管毛细管柱的富集性能进行考察,实验结果显示α酪蛋白的理论磷酸化位点覆盖率达到100%。最后,通过将所建富集和质谱鉴定方法用于小鼠肝脏全蛋白磷酸化蛋白质组的富集和分析,结果表明此功能化的开管毛细管柱可以有效用于复杂样本中磷酸化肽的富集。
[Abstract]:Reversible phosphorylation plays an important regulatory role in cell proliferation, differentiation, metabolism, intercellular communication and signal transduction pathways. Nearly 25-30% of the proteins undergo phosphorylation in their life cycle. Therefore, protein phosphorylation modification is one of the hotspots in post-translational modification proteomics. At present, mass spectrometers, including MALDI sources and ESI sources, are widely used in proteomics for their high sensitivity, accuracy and resolution. Because phosphorylated proteins are very low in vivo and easily interfered by non-phosphate peptides in mass spectrometry, the enrichment of phosphorylated peptides and phosphorylated proteins has become a key step in the analysis of phosphorylated peptides and phosphorylated proteins. At present, there are many methods and techniques for the enrichment of phosphopeptide, but there are still many problems in the application of these methods. Therefore, it is necessary to develop new enrichment methods to meet the needs of large-scale phosphorylation proteome analysis. Based on this, a new method of phosphate peptide enrichment has been developed and applied to the analysis of complex samples. The method developed in this paper is a new method for rapid enrichment of phosphopeptide by using bonded zirconium phosphate open-tube capillary column. The process is to activate the inner surface of fused quartz capillary by acid and base to make it contain free silica hydroxyl, to introduce 3-aminopropyl triethoxy silane on the surface of silica hydroxyl, and then to introduce methyl methacrylate and ethylenediamine, respectively. After three rounds of reaction, phosphoric acid group was modified on the dendritic molecule and phosphate was formed with zirconium ion. Finally, a multilayer capillary column with specific adsorption of phosphopeptide was synthesized. The enrichment conditions of the multilayer open-tube capillary column were further optimized. It was determined that when the length of the capillary column was 50cm, the injection rate of standard phosphopeptide was 0.3 渭 L/min -0.5 渭 L/min, and the enrichment effect was very good. Under the same flow rate, there is no significant difference in enrichment effect when different inner diameter multi-layer open string is used for enrichment. At the same time, the effect of better separation salt, such as ammonium chloride, on the enrichment efficiency in the separation of strong cation exchange chromatography was investigated, so that the method was compatible with the preseparation process of complex samples. After that, the loading amount and recovery rate of the capillary column were investigated by the combination of FLpTEYVATR and external standard method. The results showed that the loading amount was 48 pmoland the recovery rate was 94.1cm 脳 50 渭 m 路m ~ (-1). The selectivity and detection sensitivity of capillary column were investigated by using 伪 casein and BSA mixed enzyme digestion solution. The results showed that this method had a high selectivity for phosphopeptide and the detection limit was 10 ~ (-9) M. Under the optimized experimental conditions, the enrichment performance of bovine 伪 casein on an open-tube capillary column was investigated. The experimental results showed that the theoretical phosphorylation site coverage of 伪 casein reached 100%. Finally, the enrichment and mass spectrometry methods were applied to the enrichment and analysis of total protein phosphorylated proteome in mouse liver. The results showed that the functionalized capillary column could be effectively used for the enrichment of phosphorylated peptides in complex samples.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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