【摘要】：基質(zhì)輔助激光解析/電離質(zhì)譜(matrix-assisted laser desorption/ionization mass spectrometry,MALDI-MS)是近年發(fā)展起來(lái)的一種新型的軟電離生物質(zhì)譜,具有靈敏度高、準(zhǔn)確率高及分辨率高等特點(diǎn)。由于該質(zhì)譜分析方法優(yōu)勢(shì)明顯,早期MALDI-MS主要被用于分析質(zhì)量數(shù)較大的物質(zhì),但隨著MALDI基質(zhì)的不斷發(fā)展,其在低分子量物質(zhì)分析方面同樣受到了青睞。金屬有機(jī)骨架化合物(metal-organic frameworks,MOFs)是一類由有機(jī)小分子配體和金屬中心離子或團(tuán)簇通過(guò)金屬-配體之間的配位鍵自組裝形成的具有分子內(nèi)孔隙的新型有機(jī)-無(wú)機(jī)雜化材料,也是近二十年來(lái)在配位化學(xué)領(lǐng)域發(fā)展比較迅速,關(guān)注度比較高的新材料。由于它擁有無(wú)限的、均一的3-D拓?fù)浣Y(jié)構(gòu),所以與傳統(tǒng)的無(wú)機(jī)多孔材料相比,MOFs材料具有比表面積大、孔隙率高、結(jié)構(gòu)多樣性等優(yōu)良性質(zhì),此外它自身具有的可變的功能基團(tuán)和不飽和金屬位點(diǎn)同樣受到關(guān)注。本文利用MOFs可變功能基團(tuán)和不飽和金屬位點(diǎn)的性質(zhì),重點(diǎn)研究了MIL-101(Cr)和MIL-101(Fe)作為基質(zhì)及富集材料在MALDI-MS檢測(cè)槲皮素和磷酸化肽分析中的應(yīng)用。具體研究?jī)?nèi)容如下:MIL-101(Cr)作為MALDI-MS固體基質(zhì)靈敏的檢測(cè)槲皮素由具有不飽和金屬配位點(diǎn)的Cr3+作為金屬中心,具有π共軛結(jié)構(gòu)和紫外-可見(jiàn)光吸收性質(zhì)的對(duì)苯二甲酸作為配體合成的金屬有機(jī)骨架化合物MIL-101(Cr),其對(duì)MALDI激光能量有強(qiáng)吸收,且活化的MIL-101(Cr)具有路易斯酸性�；谏鲜鲂再|(zhì),我們選用MIL-101(Cr)作為MALDI-MS固體基質(zhì),實(shí)現(xiàn)了對(duì)黃酮類藥物小分子的無(wú)背景干擾檢測(cè),據(jù)此我們建立了靈敏檢測(cè)槲皮素的方法。在最優(yōu)條件下,該方法在0.25-7.00μg/mL的范圍內(nèi)具有良好的線性關(guān)系,檢出限為2.11 ng/mL(3σ/k),將建立的方法用于槐花中槲皮素的加標(biāo)檢測(cè),獲得了較好的回收率。Fe_3O_4/MIL-101(Fe)復(fù)合材料用于磷酸化肽富集及MALDI-MS分析由于Fe_3O_4/MIL-101(Fe)復(fù)合材料表面富含大量的不飽和配位Fe3+,結(jié)合磷酸基團(tuán)與Fe3+之間易于配位的性質(zhì),我們實(shí)現(xiàn)了復(fù)雜樣品中磷酸化肽的選擇性富集和高效分離。對(duì)于Fe_3O_4/MIL-101(Fe)復(fù)合材料,具有多孔結(jié)構(gòu)的MIL-101(Fe)可實(shí)現(xiàn)對(duì)大分子物質(zhì)的尺寸排阻,擁有磁性的Fe_3O_4納米材料可實(shí)現(xiàn)高效的磁分離。通過(guò)對(duì)標(biāo)準(zhǔn)蛋白中磷酸化肽的富集分離分析,結(jié)果表明該復(fù)合材料可對(duì)低至8 fmol的磷酸化肽實(shí)現(xiàn)富集,其富集效率達(dá)到89.8%。而對(duì)于復(fù)雜樣品體系,磷酸化肽與非磷酸化肽的濃度在1:1000時(shí),同樣可以實(shí)現(xiàn)磷酸化肽的高效分離。根據(jù)上述Fe_3O_4/MIL-101(Fe)復(fù)合材料優(yōu)良的磷酸化肽富集性質(zhì),我們對(duì)羅非魚(yú)卵蛋白中磷酸化肽進(jìn)行富集分離分析,并成功鑒定出磷酸化肽及磷酸化位點(diǎn),進(jìn)一步表明該復(fù)合材料的實(shí)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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.63;O641.4

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本文編號(hào)：2202529