二氧化鈦富集磷酸肽方法的優(yōu)化及在小鼠肝臟磷酸化蛋白質(zhì)組學研究中的應用
發(fā)布時間：2019-03-26 17:57
【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)過程，生物體通過這一過程調(diào)控多種生命活動，在哺乳動物生命過程中，非常多的蛋白會發(fā)生磷酸化修飾。不正常的磷酸化會使生命活動產(chǎn)生紊亂。 二氧化鈦富集磷酸肽法是近年來廣泛應用的一種磷酸肽富集方法，其優(yōu)點是操作簡單、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。在實際使用中影響富集效果的因素很多，對這些影響因素要分別進行優(yōu)化，才能提高富集效率。以6種標準蛋白酶切肽段混合物為研究對象，對二氧化鈦富集磷酸肽過程中的乙腈比例、三氟乙酸比例、二氧化鈦用量等條件分別進行了優(yōu)化。結果表明在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云南熱泉里分離到的厭氧、糖解、嗜熱的細菌。初步鑒定到25個磷酸化蛋白質(zhì)，為進一步研究這種極端環(huán)境下生存的低等生物的生命活動提供了參考信息。 將優(yōu)化的富集法應用于C57BL/6J小鼠肝臟蛋白磷酸化蛋白質(zhì)組研究，重點關注小鼠肝臟細胞核和細胞膜兩類具有挑戰(zhàn)性的復雜生物樣本，首先采用7cm的SDS-聚丙烯酰胺凝膠電泳預分離樣品，然后將膠切成10個條帶做膠內(nèi)酶切。在研究中，，從細胞核蛋白中共鑒定到198個磷酸化蛋白，275個非冗余磷酸化肽段，468個磷酸化位點。細胞膜中共鑒定到175個磷酸化蛋白，298個非冗余磷酸化肽段，338個磷酸化位點。
[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.
【學位授予單位】：北京工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R341
本文編號：2447788
[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.
【學位授予單位】：北京工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R341
【參考文獻】
相關期刊論文 前2條
1 遲明;畢煒;盧莊;宋麗娜;賈偉;張養(yǎng)軍;錢小紅;蔡耘;;天冬氨酸作為非特異性吸附抑制劑在二氧化鈦選擇性富集磷酸肽中的應用[J];色譜;2010年02期
2 侯春彥;馬俊鋒;陶定銀;張麗華;梁振;張玉奎;;人神經(jīng)母細胞瘤的磷酸化膜蛋白質(zhì)組分析[J];色譜;2011年09期
本文編號：2447788
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