金属有机骨架化合物在MALDI-MS检测槲皮素与磷酸化肽分析中的应用
发布时间：2018-08-25 09:55
【摘要】：基质辅助激光解析/电离质谱(matrix-assisted laser desorption/ionization mass spectrometry,MALDI-MS)是近年发展起来的一种新型的软电离生物质谱,具有灵敏度高、准确率高及分辨率高等特点。由于该质谱分析方法优势明显,早期MALDI-MS主要被用于分析质量数较大的物质,但随着MALDI基质的不断发展,其在低分子量物质分析方面同样受到了青睐。金属有机骨架化合物(metal-organic frameworks,MOFs)是一类由有机小分子配体和金属中心离子或团簇通过金属-配体之间的配位键自组装形成的具有分子内孔隙的新型有机-无机杂化材料,也是近二十年来在配位化学领域发展比较迅速,关注度比较高的新材料。由于它拥有无限的、均一的3-D拓扑结构,所以与传统的无机多孔材料相比,MOFs材料具有比表面积大、孔隙率高、结构多样性等优良性质,此外它自身具有的可变的功能基团和不饱和金属位点同样受到关注。本文利用MOFs可变功能基团和不饱和金属位点的性质,重点研究了MIL-101(Cr)和MIL-101(Fe)作为基质及富集材料在MALDI-MS检测槲皮素和磷酸化肽分析中的应用。具体研究内容如下:MIL-101(Cr)作为MALDI-MS固体基质灵敏的检测槲皮素由具有不饱和金属配位点的Cr3+作为金属中心,具有π共轭结构和紫外-可见光吸收性质的对苯二甲酸作为配体合成的金属有机骨架化合物MIL-101(Cr),其对MALDI激光能量有强吸收,且活化的MIL-101(Cr)具有路易斯酸性。基于上述性质,我们选用MIL-101(Cr)作为MALDI-MS固体基质,实现了对黄酮类药物小分子的无背景干扰检测,据此我们建立了灵敏检测槲皮素的方法。在最优条件下,该方法在0.25-7.00μg/mL的范围内具有良好的线性关系,检出限为2.11 ng/mL(3σ/k),将建立的方法用于槐花中槲皮素的加标检测,获得了较好的回收率。Fe_3O_4/MIL-101(Fe)复合材料用于磷酸化肽富集及MALDI-MS分析由于Fe_3O_4/MIL-101(Fe)复合材料表面富含大量的不饱和配位Fe3+,结合磷酸基团与Fe3+之间易于配位的性质,我们实现了复杂样品中磷酸化肽的选择性富集和高效分离。对于Fe_3O_4/MIL-101(Fe)复合材料,具有多孔结构的MIL-101(Fe)可实现对大分子物质的尺寸排阻,拥有磁性的Fe_3O_4纳米材料可实现高效的磁分离。通过对标准蛋白中磷酸化肽的富集分离分析,结果表明该复合材料可对低至8 fmol的磷酸化肽实现富集,其富集效率达到89.8%。而对于复杂样品体系,磷酸化肽与非磷酸化肽的浓度在1:1000时,同样可以实现磷酸化肽的高效分离。根据上述Fe_3O_4/MIL-101(Fe)复合材料优良的磷酸化肽富集性质,我们对罗非鱼卵蛋白中磷酸化肽进行富集分离分析,并成功鉴定出磷酸化肽及磷酸化位点,进一步表明该复合材料的实用性。
[Abstract]:Matrix assisted laser desorption / ionization mass spectrometry (matrix-assisted laser desorption/ionization mass spectrometry,MALDI-MS) is a new type of soft ionized biomass spectrum developed in recent years, which has the characteristics of high sensitivity, high accuracy and high resolution. Because of the advantage of this mass spectrum analysis method, early MALDI-MS was mainly used for the analysis of substances with large mass number, but with the development of MALDI matrix, it was also favored in the analysis of low molecular weight substances. Organometallic skeleton compounds (metal-organic frameworks,MOFs) are novel organic-inorganic hybrid materials with intramolecular pores formed by self-assembly of organic small molecular ligands and metal central ions or clusters through coordination bonds between metal-ligands. It is also a new material with rapid development and high attention in the field of coordination chemistry in recent 20 years. Because of its infinite and uniform 3-D topology, MOFs have many excellent properties, such as large specific surface area, high porosity, structural diversity and so on, compared with traditional inorganic porous materials. In addition, its own variable functional groups and unsaturated metal sites are also of concern. Based on the properties of variable functional groups and unsaturated metal sites of MOFs, the application of MIL-101 (Cr) and MIL-101 (Fe) as matrix and enrichment materials in the detection of quercetin and phosphorylated peptides by MALDI-MS was studied. The specific contents of this study are as follows: 1) the sensitive detection of quercetin by (Cr) as MALDI-MS solid matrix; Cr3 with unsaturated metal coordination point as the metal center; Terephthalic acid as ligand with 蟺 conjugated structure and UV-Vis absorption property, MIL-101 (Cr), has strong absorption of MALDI laser energy, and activated MIL-101 (Cr) has Lewis acidity. Based on the above properties, we selected MIL-101 (Cr) as MALDI-MS solid matrix to detect flavonoids small molecules without background interference. Based on this, we established a sensitive method for the detection of quercetin. Under the optimal conditions, the method has a good linear relationship in the range of 0.25-7.00 渭 g/mL, and the detection limit is 2.11 ng/mL (3 蟽 / k). The established method has been applied to the standard determination of quercetin in Sophora japonica. A better recovery rate of FeSZ / MIL-101 (Fe) composite was obtained for phosphorylated peptide enrichment and MALDI-MS analysis. Because the surface of Fe_3O_4/MIL-101 (Fe) composite was rich in unsaturated coordination Fe3, it was easy to coordinate between phosphate group and Fe3. We have achieved selective enrichment and efficient separation of phosphorylated peptides in complex samples. For Fe_3O_4/MIL-101 (Fe) composites, MIL-101 (Fe) with porous structure can eliminate the size of macromolecular materials, and Fe_3O_4 nanomaterials with magnetic properties can achieve high efficiency magnetic separation. Through the enrichment and separation of phosphorylated peptides in standard proteins, the results showed that the composite could enrich phosphorylated peptides of as low as 8 fmol, and the enrichment efficiency was 89.8%. When the concentration of phosphorylated peptide and non-phosphorylated peptide is 1: 1000, the high efficiency separation of phosphorylated peptide can also be achieved in complex sample system. According to the excellent phosphorylated peptide enrichment properties of the above Fe_3O_4/MIL-101 (Fe) composite, the phosphorylated peptides in the ovalprotein of tilapia were enriched and analyzed, and the phosphorylated peptides and phosphorylation sites were successfully identified, which further demonstrated the practicability of the composite.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.63;O641.4
[Abstract]:Matrix assisted laser desorption / ionization mass spectrometry (matrix-assisted laser desorption/ionization mass spectrometry,MALDI-MS) is a new type of soft ionized biomass spectrum developed in recent years, which has the characteristics of high sensitivity, high accuracy and high resolution. Because of the advantage of this mass spectrum analysis method, early MALDI-MS was mainly used for the analysis of substances with large mass number, but with the development of MALDI matrix, it was also favored in the analysis of low molecular weight substances. Organometallic skeleton compounds (metal-organic frameworks,MOFs) are novel organic-inorganic hybrid materials with intramolecular pores formed by self-assembly of organic small molecular ligands and metal central ions or clusters through coordination bonds between metal-ligands. It is also a new material with rapid development and high attention in the field of coordination chemistry in recent 20 years. Because of its infinite and uniform 3-D topology, MOFs have many excellent properties, such as large specific surface area, high porosity, structural diversity and so on, compared with traditional inorganic porous materials. In addition, its own variable functional groups and unsaturated metal sites are also of concern. Based on the properties of variable functional groups and unsaturated metal sites of MOFs, the application of MIL-101 (Cr) and MIL-101 (Fe) as matrix and enrichment materials in the detection of quercetin and phosphorylated peptides by MALDI-MS was studied. The specific contents of this study are as follows: 1) the sensitive detection of quercetin by (Cr) as MALDI-MS solid matrix; Cr3 with unsaturated metal coordination point as the metal center; Terephthalic acid as ligand with 蟺 conjugated structure and UV-Vis absorption property, MIL-101 (Cr), has strong absorption of MALDI laser energy, and activated MIL-101 (Cr) has Lewis acidity. Based on the above properties, we selected MIL-101 (Cr) as MALDI-MS solid matrix to detect flavonoids small molecules without background interference. Based on this, we established a sensitive method for the detection of quercetin. Under the optimal conditions, the method has a good linear relationship in the range of 0.25-7.00 渭 g/mL, and the detection limit is 2.11 ng/mL (3 蟽 / k). The established method has been applied to the standard determination of quercetin in Sophora japonica. A better recovery rate of FeSZ / MIL-101 (Fe) composite was obtained for phosphorylated peptide enrichment and MALDI-MS analysis. Because the surface of Fe_3O_4/MIL-101 (Fe) composite was rich in unsaturated coordination Fe3, it was easy to coordinate between phosphate group and Fe3. We have achieved selective enrichment and efficient separation of phosphorylated peptides in complex samples. For Fe_3O_4/MIL-101 (Fe) composites, MIL-101 (Fe) with porous structure can eliminate the size of macromolecular materials, and Fe_3O_4 nanomaterials with magnetic properties can achieve high efficiency magnetic separation. Through the enrichment and separation of phosphorylated peptides in standard proteins, the results showed that the composite could enrich phosphorylated peptides of as low as 8 fmol, and the enrichment efficiency was 89.8%. When the concentration of phosphorylated peptide and non-phosphorylated peptide is 1: 1000, the high efficiency separation of phosphorylated peptide can also be achieved in complex sample system. According to the excellent phosphorylated peptide enrichment properties of the above Fe_3O_4/MIL-101 (Fe) composite, the phosphorylated peptides in the ovalprotein of tilapia were enriched and analyzed, and the phosphorylated peptides and phosphorylation sites were successfully identified, which further demonstrated the practicability of the composite.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.63;O641.4
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