【摘要】：蛋白質(zhì)磷酸化是生物體內(nèi)一種非常重要的蛋白質(zhì)翻譯后修飾方式，磷酸化與去磷酸化是可逆的雙向調(diào)節(jié)過(guò)程，生物體通過(guò)這一過(guò)程調(diào)控多種生命活動(dòng)，在哺乳動(dòng)物生命過(guò)程中，非常多的蛋白會(huì)發(fā)生磷酸化修飾。不正常的磷酸化會(huì)使生命活動(dòng)產(chǎn)生紊亂。 二氧化鈦富集磷酸肽法是近年來(lái)廣泛應(yīng)用的一種磷酸肽富集方法，其優(yōu)點(diǎn)是操作簡(jiǎn)單、成本低。在上樣緩沖液中加入谷氨酸可以抑制非磷酸化肽段的吸附，提高富集的特異性。在實(shí)際使用中影響富集效果的因素很多，對(duì)這些影響因素要分別進(jìn)行優(yōu)化，才能提高富集效率。以6種標(biāo)準(zhǔn)蛋白酶切肽段混合物為研究對(duì)象，對(duì)二氧化鈦富集磷酸肽過(guò)程中的乙腈比例、三氟乙酸比例、二氧化鈦用量等條件分別進(jìn)行了優(yōu)化。結(jié)果表明在80%乙腈、1%三氟乙酸、40倍二氧化鈦用量（與肽段質(zhì)量比）條件下，可以取得較好的富集效果。 將優(yōu)化后的富集方法應(yīng)用于騰沖嗜熱厭氧菌磷酸化蛋白質(zhì)的分析，騰沖嗜熱厭氧菌是一種從中國(guó)云南熱泉里分離到的厭氧、糖解、嗜熱的細(xì)菌。初步鑒定到25個(gè)磷酸化蛋白質(zhì)，為進(jìn)一步研究這種極端環(huán)境下生存的低等生物的生命活動(dòng)提供了參考信息。 將優(yōu)化的富集法應(yīng)用于C57BL/6J小鼠肝臟蛋白磷酸化蛋白質(zhì)組研究，重點(diǎn)關(guān)注小鼠肝臟細(xì)胞核和細(xì)胞膜兩類(lèi)具有挑戰(zhàn)性的復(fù)雜生物樣本，首先采用7cm的SDS-聚丙烯酰胺凝膠電泳預(yù)分離樣品，然后將膠切成10個(gè)條帶做膠內(nèi)酶切。在研究中，，從細(xì)胞核蛋白中共鑒定到198個(gè)磷酸化蛋白，275個(gè)非冗余磷酸化肽段，468個(gè)磷酸化位點(diǎn)。細(xì)胞膜中共鑒定到175個(gè)磷酸化蛋白，298個(gè)非冗余磷酸化肽段，338個(gè)磷酸化位點(diǎn)。
[Abstract]:Protein phosphorylation is a very important post-translational modification of proteins in organisms. Phosphorylation and dephosphorylation are reversible two-way regulation processes through which organisms regulate a variety of life activities in mammalian life. A lot of proteins are phosphorylated. Abnormal phosphorylation can derail the activity of life. Titanium dioxide-enriched phosphopeptide method is a widely used phosphate peptide enrichment method in recent years, which has the advantages of simple operation and low cost. Glutamic acid can inhibit the adsorption of non-phosphorylated peptides and increase the specificity of enrichment. There are many factors that affect the enrichment effect in practical application, and these factors should be optimized separately to improve the enrichment efficiency. Six kinds of standard protease-cleaving peptide mixtures were used to optimize the conditions of acetonitrile ratio, trifluoroacetic acid ratio and TIO _ 2 dosage during the enrichment of phosphopeptide by TIO _ 2. The results showed that under the conditions of 80% acetonitrile, 1% trifluoroacetic acid and 40 times the amount of titanium dioxide (to the peptide mass ratio), the better enrichment effect could be obtained. The optimized enrichment method was applied to the analysis of phosphorylated proteins in Tengchong thermophilic anaerobic bacteria, which is a kind of anaerobic, glycolytic and thermophilic bacteria isolated from hot spring of Yunnan Province, China. Twenty-five phosphorylated proteins were identified preliminarily, which provided reference information for further study of the life activities of low-class organisms living in this extreme environment. The optimized enrichment method was applied to protein phosphorylation proteome study in the liver of C57BL/6J mice, focusing on the challenging and complex biological samples of mouse liver nuclei and cell membranes. 7cm's SDS- polyacrylamide gel electrophoresis was used to pre-isolate the samples, and then the gel was cut into 10 bands and digested in the gel. In this study, 198 phosphorylated proteins, 275 non-redundant phosphorylated peptides and 468 phosphorylation sites were identified from the nuclear proteins. 175 phosphorylated proteins, 298 non-redundant phosphorylated peptides and 338 phosphorylation sites were identified.
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R341

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