C18固相萃取小柱样品处理方法优化及冠心病尿液差异蛋白质组学分析

发布时间：2018-05-10 07:32

本文选题：反相色谱 + C18固相萃取小柱　；参考：《北京协和医学院》2015年硕士论文

【摘要】：研究背景与目的：针对少量且复杂蛋白质组样品,开发一种耗时短,操作简便的预处理方法。研究方法：以人的脑海马组织蛋白为研究对象,使用反相C18固相萃取小柱进行样品分离,并与反相高效液相色谱法进行比较分析。根据四种乙腈梯度设置方案的分离效果以确定最佳的洗脱条件。研究结果：八种乙腈浓度洗脱仅耗时10 min,为反相高效液相色谱法的1/4,但鉴定蛋白总数占高效液相色谱法的85.5%。乙腈洗脱浓度为5%,15%,20%,90%时分离30μg人的海马多肽混合物可以得到较优的分离效果,两次重复实验鉴定蛋白重复率达到70%。研究结论：该方法操作易行,省时,成本低,是进行蛋白质组学分析中少量样品的一个简便预处理方法。研究背景与目的：针对尿液样品有易获取,处理方法简便的优点,近年来尿蛋白质组学引起人们广泛关注。以往动脉粥样硬化的尿蛋白质组学研究主要应用毛细管电泳-质谱联用技术,报道多种多肽标记物。本文从尿液蛋白质组学入手,目的在于发现动脉粥样硬化疾病在蛋白水平的表达差异。研究方法：采用iTRAQ (Isobaric tags for relative and absolute quantitation)定量标记串联质谱分析技术从动脉粥样硬化患者及对照组尿液样品中发现差异表达蛋白。研究结果：经过质谱分析,Mascot 及 Scaffold数据库检索从尿液中共鉴定856个蛋白,其中定量蛋白737个。动脉粥样硬化患者及对照组之间尿液中存在90个差异表达蛋白,倍数变化设置1.5倍。经过IPA功能分析发现差异表达蛋白与糖类,脂类代谢及炎症反应密切相关。研究结论：尿液是动脉粥样硬化疾病标记物发现的理想来源。尿液中差异蛋白表达表达情况可以反映动脉粥样硬化疾病状态下细胞内外的病理生理变化。
[Abstract]:Background & AIM: to develop a simple and time consuming pretreatment method for a small amount of complex proteome samples. Methods: the human brain tissue protein was separated by reverse phase C 18 solid phase extraction column, and compared with that of the reverse phase high performance liquid chromatography (RP-HPLC). According to the separation effect of four acetonitrile gradient setting schemes, the best elution conditions were determined. The results showed that the elution time of eight acetonitrile concentrations was only 10 min, which was 1 / 4 of that by RP-HPLC, but the total number of identified proteins was 85.5% of that by HPLC. The separation of 30 渭 g human hippocampal polypeptide mixture with acetonitrile elution concentration was 20% and 90 渭 g, and the protein repetition rate reached 70%. Conclusion: this method is easy to operate, time saving and low cost. It is a simple pretreatment method for a small amount of samples in proteomics analysis. Background & AIM: because urine samples are easy to be obtained and processed, proteomics of urine has attracted more and more attention in recent years. In the past, urinary proteomics of atherosclerosis was mainly reported by capillary electrophoresis-mass spectrometry. The aim of this paper is to find out the difference of protein expression in atherosclerotic diseases. Methods: the differentially expressed proteins were found in urine samples from patients with atherosclerosis and control group by iTRAQ tags for relative and absolute quantitative tandem mass spectrometry. Results: a total of 856 proteins, including 737 quantitative proteins, were identified from urine by Mascot and Scaffold database. There were 90 differentially expressed proteins in urine between atherosclerotic patients and controls. IPA functional analysis showed that differentially expressed proteins were closely related to carbohydrate, lipid metabolism and inflammation. Conclusion: urine is an ideal source for the discovery of markers for atherosclerosis. The expression of differential protein in urine can reflect the pathophysiological changes of cells in atherosclerotic state.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.4

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