二氧化钛富集磷酸肽方法的优化及在小鼠肝脏磷酸化蛋白质组学研究中的应用
发布时间：2019-03-26 17:57
【摘要】：蛋白质磷酸化是生物体内一种非常重要的蛋白质翻译后修饰方式，磷酸化与去磷酸化是可逆的双向调节过程，生物体通过这一过程调控多种生命活动，在哺乳动物生命过程中，非常多的蛋白会发生磷酸化修饰。不正常的磷酸化会使生命活动产生紊乱。 二氧化钛富集磷酸肽法是近年来广泛应用的一种磷酸肽富集方法，其优点是操作简单、成本低。在上样缓冲液中加入谷氨酸可以抑制非磷酸化肽段的吸附，提高富集的特异性。在实际使用中影响富集效果的因素很多，对这些影响因素要分别进行优化，才能提高富集效率。以6种标准蛋白酶切肽段混合物为研究对象，对二氧化钛富集磷酸肽过程中的乙腈比例、三氟乙酸比例、二氧化钛用量等条件分别进行了优化。结果表明在80%乙腈、1%三氟乙酸、40倍二氧化钛用量（与肽段质量比）条件下，可以取得较好的富集效果。 将优化后的富集方法应用于腾冲嗜热厌氧菌磷酸化蛋白质的分析，腾冲嗜热厌氧菌是一种从中国云南热泉里分离到的厌氧、糖解、嗜热的细菌。初步鉴定到25个磷酸化蛋白质，为进一步研究这种极端环境下生存的低等生物的生命活动提供了参考信息。 将优化的富集法应用于C57BL/6J小鼠肝脏蛋白磷酸化蛋白质组研究，重点关注小鼠肝脏细胞核和细胞膜两类具有挑战性的复杂生物样本，首先采用7cm的SDS-聚丙烯酰胺凝胶电泳预分离样品，然后将胶切成10个条带做胶内酶切。在研究中，，从细胞核蛋白中共鉴定到198个磷酸化蛋白，275个非冗余磷酸化肽段，468个磷酸化位点。细胞膜中共鉴定到175个磷酸化蛋白，298个非冗余磷酸化肽段，338个磷酸化位点。
[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.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341
本文编号：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.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341
【参考文献】
相关期刊论文 前2条
1 迟明;毕炜;卢庄;宋丽娜;贾伟;张养军;钱小红;蔡耘;;天冬氨酸作为非特异性吸附抑制剂在二氧化钛选择性富集磷酸肽中的应用[J];色谱;2010年02期
2 侯春彦;马俊锋;陶定银;张丽华;梁振;张玉奎;;人神经母细胞瘤的磷酸化膜蛋白质组分析[J];色谱;2011年09期
本文编号：2447788
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