本文選題：多層開(kāi)管毛細(xì)管柱 + 磷酸鋯　； 參考：《安徽醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 可逆的磷酸化過(guò)程在細(xì)胞增殖、分化、代謝、細(xì)胞間通訊以及信號(hào)轉(zhuǎn)導(dǎo)通路等重要的生物學(xué)過(guò)程中發(fā)揮著重要的調(diào)節(jié)作用,真核細(xì)胞中,有近25-30%的蛋白在其生活史中會(huì)經(jīng)歷磷酸化過(guò)程。因此,蛋白質(zhì)的磷酸化修飾是翻譯后修飾蛋白質(zhì)組研究中的熱點(diǎn)之一。目前,包括MALDI源和ESI源在內(nèi)的質(zhì)譜儀以其高靈敏度、準(zhǔn)確度和分辨率而被廣泛用于蛋白質(zhì)組學(xué)研究中。由于磷酸化蛋白在生物體內(nèi)含量很低,且在質(zhì)譜分析時(shí)易受到非磷酸肽的干擾,因而磷酸肽和磷酸化蛋白質(zhì)的富集成為磷酸化肽段和磷酸化蛋白質(zhì)分析的一個(gè)關(guān)鍵步驟。目前已經(jīng)有很多富集磷酸肽的方法和技術(shù),但這些方法在應(yīng)用時(shí)仍然存在不少問(wèn)題,因而需要發(fā)展新的富集方法,以滿(mǎn)足大規(guī)模磷酸化蛋白質(zhì)組分析的需要�；诖�,本文開(kāi)展了一種磷酸肽富集的新方法研究,并將其應(yīng)用于復(fù)雜樣品的分析。 本課題發(fā)展的方法為一種利用鍵合磷酸鋯開(kāi)管毛細(xì)管柱的制備及用于快速富集磷酸肽的新方法。其過(guò)程是通過(guò)酸堿活化熔融石英毛細(xì)管內(nèi)表面,使其帶有自由的硅羥基,在硅羥基表面引入3-氨丙基三乙氧基硅烷,之后再分別引入甲基丙烯酸甲酯和乙二胺,如此進(jìn)行三輪反應(yīng)后,在形成的樹(shù)枝狀大分子表面進(jìn)行磷酸基團(tuán)修飾并與鋯離子生成磷酸鹽,最終合成具有特異性吸附磷酸肽分子的多層開(kāi)管毛細(xì)管柱。 進(jìn)一步對(duì)該多層開(kāi)管毛細(xì)管柱的富集條件進(jìn)行了優(yōu)化,確定出當(dāng)開(kāi)管毛細(xì)管柱長(zhǎng)度為50 cm時(shí),標(biāo)準(zhǔn)磷酸肽的進(jìn)樣流速在0.3μL/min -0.5μL/min時(shí),均有很好的富集效果。并且在同樣的流速條件下,使用不同內(nèi)徑的多層開(kāi)管柱進(jìn)行富集時(shí),富集效果沒(méi)有顯著性差異。同時(shí)還考察了強(qiáng)陽(yáng)離子交換色譜分離中分離效果較好的鹽,如氯化銨對(duì)富集效果的影響,以便該方法與復(fù)雜樣本的預(yù)分離過(guò)程相兼容。之后,還用合成的磷酸肽段(FLpTEYVATR)與外標(biāo)法相結(jié)合,對(duì)開(kāi)管毛細(xì)管柱的負(fù)載量和回收率等進(jìn)行考察,確定出負(fù)載量為48 pmol,回收率為94.1%(50 cm×50μm)。利用α酪蛋白與BSA混合酶切溶液,考察了毛細(xì)管開(kāi)管柱的選擇性和檢測(cè)靈敏度,結(jié)果表明此方法對(duì)磷酸肽有較高的選擇性,且檢測(cè)限達(dá)到10-9M。在優(yōu)化的實(shí)驗(yàn)條件下,利用牛α酪蛋白對(duì)開(kāi)管毛細(xì)管柱的富集性能進(jìn)行考察,實(shí)驗(yàn)結(jié)果顯示α酪蛋白的理論磷酸化位點(diǎn)覆蓋率達(dá)到100%。 最后,通過(guò)將所建富集和質(zhì)譜鑒定方法用于小鼠肝臟全蛋白磷酸化蛋白質(zhì)組的富集和分析,結(jié)果表明此功能化的開(kāi)管毛細(xì)管柱可以有效用于復(fù)雜樣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 偽 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 偽 casein on an open-tube capillary column was investigated. The experimental results showed that the theoretical phosphorylation site coverage of 偽 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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R341

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本文編號(hào)：1920978