内部萃取电喷雾电离质谱技术在组织样品分析中的应用研究

发布时间：2018-05-19 12:21

本文选题：质谱 + 内部萃取电喷雾电离　；参考：《东华理工大学》2017年硕士论文

【摘要】：质谱技术具有灵敏度高、分析速度快、能提供分子结构信息等特点,在生命科学研究领域扮演着重要角色,常用于组织样品分析。传统地,组织样品分析一般需经过研磨、萃取、分离等繁杂的样品预处理过程,导致耗时低效,无法满足大量样品高通量分析的实际需求,且不可避免地使组织样品中的一些活性成分损失。近年来,随着新兴常压质谱技术的发展,使得组织样品可在无需样品预处理的条件下进行实时、非破坏、在线直接质谱分析,大大提高了分析效率。本文着重介绍了一种新型常压质谱技术,内部萃取电喷雾电离质谱(iEESI-MS)技术在植物组织、动物组织和人体组织分析中的应用研究,并简要展望了该技术在植物代谢组学、食品科学、临床医学等领域的发展趋势。基于对传统质谱技术在组织样品分析中应用研究的简要概述,本论文的主要研究内容如下:1.iEESI-MS技术在植物组织分析中的应用研究。采用iEESI-MS技术对芦笋新鲜茎块和干燥芦笋茎块、不同纳米材料培养绿豆芽的根、茎和子叶组织及不同产地脐橙内表皮组织与果肉组织进行了直接质谱分析。1)对芦笋新鲜茎块和干燥芦笋茎块进行直接分析,结合碰撞诱导解离(CID)实验,鉴定了对药物和自然产品研发具有重要意义的多种代表性营养成分。该结果不仅为食源性植物组织样品的快速分析提供了一种质谱分析新方法,而且对农业的发展具有一定的指导意义。2)对不同纳米材料包括碲化镉量子点(CdTe QDs)、纳米金(Au NPs)和纳米银(Ag NPs)及水培养绿豆芽的根、茎和子叶组织进行直接质谱分析,结果表明纳米材料会对绿豆芽的能量代谢过程(如糖类物质的转化、分解等)产生一定的影响。该工作从分子层面上研究了纳米材料对绿豆芽代谢产生的影响,为探究由纳米材料引起的其它效应如细胞凋亡或坏死等奠定了一定的基础。3)对不同产地脐橙的内果皮组织和果肉组织进行直接iEESI-MS分析,结果表明脐橙果肉组织与内果皮组织中的化学成分既存在相似性,也具有特异性。结合主成分分析(PCA),使不同产地脐橙之间的品质差异直观地表现了出来。这进一步说明iEESI-MS技术可直接获得不同品质脐橙在分子水平上的差异,有望为判断其它果蔬品质的优劣提供参考。2.iEESI-MS技术在动物组织分析中的应用。采用iEESI-MS技术对不同肉质品中痕量瘦肉精成分进行了定性、定量分析,并研究了瘦肉精代谢对猪肉品质的影响。1)利用iEESI-MS技术从分子层面上成功地实现了对不同肉质品(包括猪肉、羊肉、牛肉等)中常见痕量瘦肉精成分的高通量筛查,结果表明iEESI-MS技术可快速检测到不同肉质品中的痕量瘦肉精成分,具有样品耗量低、操作简便、分析速度快等优点,为打击含瘦肉精的劣质肉品及杜绝运动员食源性兴奋剂超标提供了新的科学工具。2)利用iEESI-MS技术和专用精准定量取样装置,对猪肉样品中的6种瘦肉精包括沙丁胺醇、克伦特罗、莱克多巴胺、妥布特罗、特布他林和溴布特罗进行直接定量分析。实验结果表明,6种瘦肉精在0.01-1000μg/kg浓度范围内,具有较好的对数型线性关系;检出限(LOD)为0.002μg/kg,相对标准偏差(RSD)在3.2-11.8%之间。通过国标方法气相-质谱(GC-MS)技术对iEESI-MS结果进行验证,表明两者结果接近度在91-95%之间。该工作进一步将常压质谱技术对待测物进行定量分析的能力从固态样品的表面(以μg/cm2为单位)延伸到整体组织样品内部(以μg/kg为单位)。3)利用iEESI-MS技术分析了含克伦特罗、沙丁胺醇及正常猪肉在营养成分之间的差别,结合相关性分析和PCA,找到了多种与猪肉品质相关的生物标志物分子,发现瘦肉精代谢可能影响了线粒体内膜外存在的调控L-肉碱与脂肪酸结合的肉碱脂酰转移酶I的生物活性,使脂肪酸进入线粒体基质的过程受到了影响,从而干扰了脂肪酸合成磷脂酰胆碱的过程。3.iEESI-MS技术在人体组织分析中的应用研究。1)采用iEESI-MS技术,利用21种不同组成的CH3OH/H2O为萃取溶剂,对肺癌癌症组织与癌旁组织进行直接质谱分析,结果表明CH3OH:H2O=30:70(v/v)是适合肺癌组织样品内部磷脂类物质萃取的最佳溶剂条件。2)在前期最佳优化实验条件下,对肺癌癌症组织与癌旁组织进行iEESI-MS分析,结果表明iEESI-MS技术可快速识别癌症组织与癌旁组织在分子层次的差异。结合PCA找到了对区分癌症组织与癌旁组织有显著贡献的磷脂类物质等生物标志物分子,为进一步探究磷脂类物质在癌症发病机制中的作用、术中准确判定癌症边界、确保恶性肿瘤完全切除等提供参考依据。
[Abstract]:Mass spectrometry has the characteristics of high sensitivity, fast analysis and providing information on molecular structure. It plays an important role in the field of life science. It is often used in the analysis of tissue samples. Traditionally, tissue sample analysis usually needs to be pretreated with complex samples such as grinding, extraction and separation, which results in low efficiency and can not satisfy a large number of samples. The actual demand for high flux analysis and the loss of some active components in the tissue samples is unavoidable. In recent years, with the development of the emerging atmospheric mass spectrometry technology, the tissue samples can be carried out in real time, non destructive and on-line direct qualitative analysis without sample pretreatment. This paper greatly improves the efficiency of analysis. A new type of atmospheric pressure mass spectrometry, the application of internal extraction electrospray ionization mass spectrometry (iEESI-MS) technology in plant tissue, animal tissue and human tissue analysis, is studied. The development trend of this technology in plant metabolism, food science, clinical medicine and other fields is briefly prospected. Based on the analysis of traditional mass spectrometry technology in tissue samples The main contents of this study are as follows: the main contents of this thesis are as follows: the application of 1.iEESI-MS technology in the analysis of plant tissue. Using iEESI-MS technology, the fresh stem block and dry asparagus stem block of asparagus, the root, stem and cotyledon tissue of different nanomaterials and the inner skin tissue of navel orange from different producing areas and flesh tissue are introduced. Direct mass spectrometry (.1) was used to direct analysis of fresh asparagus stem block and dry asparagus stem block. Combined with collision induced dissociation (CID) experiment, a variety of representative nutrients were identified for the research and development of drugs and natural products. This result not only provides a kind of mass spectrometric analysis for the rapid analysis of food borne plant tissue samples. The new method has a certain guiding significance for the development of agriculture.2) the direct mass spectrometric analysis of the root, stem and cotyledons of different nanomaterials, including cadmium telluride quantum dots (CdTe QDs), nanoscale gold (Au NPs) and nano silver (Ag NPs) and water culture green bean sprouts, indicates that the nanomaterials can metabolize the energy of mung bean sprouts (such as sugars). The effect of nanomaterials on the metabolism of mung bean sprouts was studied at the molecular level, and a basis for exploring other effects, such as cell apoptosis or necrosis, caused by nanomaterials, such as cell apoptosis or necrosis, was studied at the molecular level, and the direct iEES of the inner pericarp tissue and flesh tissue of navel orange from different origins was carried out. The results of I-MS analysis showed that the chemical components in the flesh and the endocarp tissues of the navel orange were both similar and specific. Combining with principal component analysis (PCA), the quality differences between navel oranges in different habitats were displayed intuitively. This further illustrates that iEESI-MS technology can directly obtain the difference in molecular level of different quality navel orange. The application of reference.2.iEESI-MS technology in animal tissue analysis was provided to judge the quality of other fruits and vegetables. IEESI-MS technology was used to determine the trace amount of lean meat in different meat quality, quantitative analysis, and study the effect of the metabolism of lean meat on the quality of pork.1) using iEESI-MS technology to be successful at the molecular level. The high throughput screening of trace lean meat ingredients in different meat products (including pork, mutton, beef and so on) was carried out. The results showed that the iEESI-MS technique could quickly detect the trace content of lean meat in different meat products. It has the advantages of low sample consumption, simple operation and fast analysis, which is to combat the inferior meat and Du with lean meat essence. A new scientific tool,.2, a new scientific tool for athlete food borne stimulants, is provided by.2) using iEESI-MS technology and special precision quantitative sampling device to direct quantitative analysis of 6 kinds of lean meat concentrate in pork samples including salbutamol, clenbuterol, ractopamine, Butero, terbutaline and Bbu Tero. The experimental results show that 6 kinds of lean meat essence In the concentration range of 0.01-1000 mu g/kg, there is a better logarithmic linear relationship; the detection limit (LOD) is 0.002 mu g/kg and the relative standard deviation (RSD) is between 3.2-11.8%. The results of iEESI-MS are verified by the national standard method gas phase mass spectrometry (GC-MS) technology, indicating that the results are close to 91-95%. This work further applies the atmospheric mass spectrometry technology. The capacity for quantitative analysis of the samples was extended from the surface of the solid sample (with mu g/cm2) to the whole tissue sample (g/kg unit).3) and the difference between the nutrients containing clenbuterol, salbutamol and normal pork was analyzed by iEESI-MS technology, and a variety of pork products were found in combination with the correlation analysis and PCA. The biomarkers of qualitative related biomarkers found that the metabolism of lean meat sperm may affect the biological activity of the extracellular matrix of L- carnitine and fatty acids, which regulates the presence of the mitochondria in the mitochondria, and affects the process of the fatty acids entering the mitochondrial matrix, which interferes with the.3.iEESI-MS technology of fatty acids in the synthesis of phosphatidylcholine. Application of.1) in human tissue analysis (.1) using iEESI-MS technology and using 21 different components of CH3OH/H2O as extraction solvent to analyze lung cancer tissues and adjacent tissues by direct mass spectrometry. The results show that CH3OH:H2O=30:70 (v/v) is the best solvent condition for the extraction of phosphor substances in lung cancer tissue samples,.2) at the earliest stage. IEESI-MS analysis of cancer tissues and para cancerous tissues in lung cancer showed that iEESI-MS technology could quickly identify the difference between cancer tissue and para cancer tissue at the molecular level. Combined with PCA, the biomarker molecules, such as phosphor substances, which have significant contributions to the differentiation of cancer tissues and adjacent tissues, were found. To explore the role of phospholipids in the pathogenesis of cancer, to provide accurate evidence for accurate diagnosis of cancer boundaries and to ensure complete resection of malignant tumors.
【学位授予单位】：东华理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q503

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