受精15.5天胎鼠血清和新生鼠血清蛋白质组学比较研究
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摘要
血液是人体的重要体液,包含了来自全身各组织分泌的蛋白和细胞死亡渗出的蛋白,在某种程度上可以说血液是人体的窗口。在胎儿发育过程中,尤其是胎儿发育中后期,各器官无论在形态还是在功能都发生了巨大的变化。基于液质联用的蛋白质组学分析技术能对蛋白质——生命功能的具体执行者,进行高通量、大规模的全面研究。通过使用液质联用技术对不同时期胎鼠血清进行蛋白质组学鉴定分析,能够更好的了解生命发育的过程。
考虑到胎鼠血清个体差异,分别对受精15.5天胎鼠和新生鼠血清的个体样本和混合样本进行质谱鉴定。以混合样本三次质谱鉴定的变异系数(作为实验方法的差异)为对照,计算个体样本变异系数与混合样本变异系数的比值,获得每个蛋白在去除质谱仪器等实验误差造成的影响后,在蛋白质组学水平上进行个体差异性评价。
应用毛细管液相色谱(反相)和质谱联用方法,在质谱鉴定可信度为99%的条件下,在个体样本中,共鉴定的669个蛋白,对鉴定的蛋白进行双侧T检验,在95%的可信区间内分别有35个蛋白显著性上升和58个蛋白显著性降低。对鉴定的蛋白进行功能注释,并对在血浆中参与运输、防御、酶活性调节、凝血等生物学过程的蛋白进行分析,共发现9种补体分子,均上升,其中6种出现显著性上升；酶活性调节物分子中4种显著性下降,13种显著性上升；载脂蛋白分子显著减少,11种载脂蛋白分子中8种显著性下降,1种显著性升高；同时发现不同种类血红蛋白分子出现改变。
蛋白翻译后修饰对蛋白结构和功能起着重要的调节作用。通过对不同时期血清混合样本蛋白的翻译后修饰的研究,发现了50种不同的修饰。通过对修饰位点的定量比较,发现受精15.5天和新生鼠血清蛋白翻译后修饰的状态不同,为以后研究翻译后修饰在血清发育中的作用提供线索。
Blood, as the most important body fluid, contains lots of proteins secreted by organs or leaked from dead cells. To some extent, blood is a window of the body. During the fetal development, especially the midanaphase, each organ changes a lot in morphous and functions. Based on LC-MS/MS high throughput proteomics technologies, proteins as function performer are investagated. Analysing plasma or serum proteome of different period in fetal development, may have a better understanding biology of life.
Considering the diversity of rat fetus in blood, pooled serum specimens and individual serum specimens from E15.5fetus and newborn rat were collected and analyzed using LC-MS/MS technology. Using three times LC-MS/MS analysis of same pooled specimens as instrumental variance control, The ratio of individual samples' coefficient variation vs pooled samples'coefficient variation were used to reflect the diversity of individual samples in proteomics level.
Using LC-MS/MS,669proteins were identified in the individual specimens with the confidence of99%. After two tailed T test, with95%confidence, from15.5d fetus to new born rat,35proteins increased and58decreased significantly. The functional analysis reveals that all9identified complement molecules increased, and6increased significantly.13enzyme regulators increased while4decreased significantly,8of11apolipoproteins decreased significantly. Hemoglobin species pattern were different at the two stages.
Protein modifications can change the protein's structure and functions. We found50different modifications in pooled specimens of the two stages. Some proteins are differentially modifid. This provides clues for the studying function of the modificaion in these serum proteins in the future.
考虑到胎鼠血清个体差异,分别对受精15.5天胎鼠和新生鼠血清的个体样本和混合样本进行质谱鉴定。以混合样本三次质谱鉴定的变异系数(作为实验方法的差异)为对照,计算个体样本变异系数与混合样本变异系数的比值,获得每个蛋白在去除质谱仪器等实验误差造成的影响后,在蛋白质组学水平上进行个体差异性评价。
应用毛细管液相色谱(反相)和质谱联用方法,在质谱鉴定可信度为99%的条件下,在个体样本中,共鉴定的669个蛋白,对鉴定的蛋白进行双侧T检验,在95%的可信区间内分别有35个蛋白显著性上升和58个蛋白显著性降低。对鉴定的蛋白进行功能注释,并对在血浆中参与运输、防御、酶活性调节、凝血等生物学过程的蛋白进行分析,共发现9种补体分子,均上升,其中6种出现显著性上升；酶活性调节物分子中4种显著性下降,13种显著性上升；载脂蛋白分子显著减少,11种载脂蛋白分子中8种显著性下降,1种显著性升高；同时发现不同种类血红蛋白分子出现改变。
蛋白翻译后修饰对蛋白结构和功能起着重要的调节作用。通过对不同时期血清混合样本蛋白的翻译后修饰的研究,发现了50种不同的修饰。通过对修饰位点的定量比较,发现受精15.5天和新生鼠血清蛋白翻译后修饰的状态不同,为以后研究翻译后修饰在血清发育中的作用提供线索。
Blood, as the most important body fluid, contains lots of proteins secreted by organs or leaked from dead cells. To some extent, blood is a window of the body. During the fetal development, especially the midanaphase, each organ changes a lot in morphous and functions. Based on LC-MS/MS high throughput proteomics technologies, proteins as function performer are investagated. Analysing plasma or serum proteome of different period in fetal development, may have a better understanding biology of life.
Considering the diversity of rat fetus in blood, pooled serum specimens and individual serum specimens from E15.5fetus and newborn rat were collected and analyzed using LC-MS/MS technology. Using three times LC-MS/MS analysis of same pooled specimens as instrumental variance control, The ratio of individual samples' coefficient variation vs pooled samples'coefficient variation were used to reflect the diversity of individual samples in proteomics level.
Using LC-MS/MS,669proteins were identified in the individual specimens with the confidence of99%. After two tailed T test, with95%confidence, from15.5d fetus to new born rat,35proteins increased and58decreased significantly. The functional analysis reveals that all9identified complement molecules increased, and6increased significantly.13enzyme regulators increased while4decreased significantly,8of11apolipoproteins decreased significantly. Hemoglobin species pattern were different at the two stages.
Protein modifications can change the protein's structure and functions. We found50different modifications in pooled specimens of the two stages. Some proteins are differentially modifid. This provides clues for the studying function of the modificaion in these serum proteins in the future.
引文
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31. Veenstra TD:Global and targeted quantitative proteomics for biomarker discovery. J Chromatogr B Analyt Technol Biomed Life Sci 2007,847(1):3-11.
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