Schiff碱类化合物作为MALDI质谱基质在负离子模式下检测小分子分析物的效果评价
发布时间:2018-11-06 11:21
【摘要】:基质辅助激光解吸离子化质谱(MALDI-MS)以其软电离的特点,能够获得较完整的分子离子信息,常用于生物大分子如蛋白、核酸和合成聚合物的分析。同时,由于样品准备简单,分析速度快,灵敏度高,样品消耗少,耐盐性好等诸多优点,在分析实际样品中动植物代谢物和农药残留等小分子样品也有着不可替代的优势。然而,在实际MALDI-MS检测过程中经常需要使用基质,传统基质在小分子区域产生基质离子峰,严重干扰小分子分析物的测定,而且分析物信号强度的重现性较差,不利于其在小分子定量分析中的应用。因此,本文开发了一种新的Schiff碱类化合物,并将其用作MALDI基质,在负离子模式下,成功实现了对小分子分析物的检测,实验内容主要包含以下三个方面:(1)使用苯胺和醛类物质一步脱水缩合反应,合成了六种Schiff碱类化合物(S1、S2、S3、S4、S5和S6),将其用作MALDI基质在负离子模式下分析了脂肪酸类化合物,评价了基质的测试效果。使用紫外吸收光谱和量子化学计算的方法探讨了不同Schiff碱基质产生不同检测效果的原因。经过优化,最终选取S1作为MALDI基质分别检测了脂肪酸标准品和血清中的游离脂肪酸。使用液液萃取的方法对血清中脂肪酸进行粗提取。采取加入内标(C17:1)和种子法点样(提高基质与分析物共结晶的均匀度)两种方法来提高定量分析的重现性,最终实现了人血清中游离脂肪酸的定量分析。结果显示:使用Schiff碱S1基质定性定量分析脂肪酸具有无基质干扰、高通量、高灵敏度、分离步骤简单、无需衍生化、需样量少等优点。(2)考察了Schiff碱S1作为MALDI基质在负离子模式下检测其它酸性及醇类小分子的通用性。使用S1基质检测了六种氨基酸(色氨酸、赖氨酸、组氨酸、苏氨酸、亮氨酸、谷氨酸)、两种磷脂(1,2-二油酰-sn-甘油-3磷脂和鞘磷脂)、四种药物小分子(炔雌醇、青蒿素、阿霉素和地塞米松)以及单宁酸分子,均可获得目标分析物的质谱峰。检测不同氨基酸分子时,均能得到氨基酸失去一个质子的准分子离子峰,但各氨基酸的检测信噪比有所不同,通过量子化学计算,我们发现电离能较低的氨基酸检测效果较好,信噪比高。对于磷脂的检测,分析物信噪比高,谱图干净无基质干扰。我们对分析物碎裂峰的来源进行了机理讨论及验证。我们的结果进一步显示:Schiff碱S1作为MALDI基质在负离子模式下,对于酸性小分子、脂类及醇类物质具有一定的通用性。(3)据文献报道,化合物9AA、DMAN和DPN也可作为MALDI基质在负离子模式下使用,检测小分子分析物具有基质干扰少的优点。我们通过以下几个实验将Schiff碱S1基质与这些碱性基质进行了比较:1)基质与样品不同摩尔比下对样品峰强的影响;2)基质真空下的稳定性;3)在不同浓度下检测分析物时基质峰的干扰;4)基质的固体紫外实验;5)使用量子化学手段,计算各碱性基质在气相与液相中分别对酸性小分子的羧基H-O键长的影响以及这些碱性基质在气相中的质子亲和能(PA)、去质子的质子亲和能(PA[M-H]-)、电离能(IE)和电子亲和能(EA)等。实验结果显示:Schiff碱S1作为基质具有真空下稳定、利于定量分析、灵敏度高等优势。在这篇论文中,我们报道了一种新开发的Schiff碱类基质化合物,在MALDI负离子模式下可有效减少基质对小分子分析物的干扰,对酸性,醇类和磷脂等多种化合物实现简单、快速、准确地定性定量分析。我们结合实验结果和量子化学手段阐述了基质可能的作用机理。
[Abstract]:The matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is characterized by its soft ionization and can be used in the analysis of biological macromolecules such as proteins, nucleic acids and synthetic polymers. At the same time, as the sample preparation is simple, the analysis speed is high, the sensitivity is high, the sample consumption is low, the salt tolerance is good, and the like, and the small molecular sample such as the animal and plant metabolites and the pesticide residue in the actual sample also has an irreplaceable advantage. However, in the actual MALDI-MS detection process, it is often necessary to use the matrix, the traditional matrix produces the matrix ion peak in the small molecule region, the measurement of the small molecular analyte is seriously disturbed, and the reproducibility of the signal intensity of the analyte is poor, which is not conducive to the application in the quantitative analysis of small molecules. Therefore, a new Schiff base compound is developed and used as a MALDI matrix. In the negative ion mode, the detection of small molecular analytes has been successfully achieved. The experimental contents mainly include the following three aspects: (1) a one-step dehydration condensation reaction using aniline and aldehyde substances, Six Schiff base compounds (S1, S2, S3, S4, S5 and S6) were synthesized and used as the MALDI matrix to analyze the fatty acid compounds in the negative ion mode, and the test results of the matrix were evaluated. The reasons for different detection effects of different Schiff bases were discussed using the method of ultraviolet absorption spectrum and quantum chemical calculation. The free fatty acids in the fatty acid standard and the serum are respectively detected by the S1 as the MALDI matrix through the optimization. The fatty acid in serum was extracted by liquid-liquid extraction. The reproducibility of quantitative analysis was improved by adding internal standard (C17: 1) and seed method (improving the uniformity of the co-crystallization of matrix and analyte), and the quantitative analysis of free fatty acids in human serum was achieved. The results show that using the Schiff base S1 matrix to qualitatively and quantitatively analyze the fatty acid has the advantages of no matrix interference, high flux, high sensitivity, simple separation step, no need of derivatization, less sample amount, and the like. (2) The versatility of Schiff base S1 as MALDI matrix in the detection of other acid and alcohol molecules in negative ion mode was investigated. Six kinds of amino acids (tryptophan, lysine, histidine, threonine, leucine, glutamic acid), two kinds of phospholipids (1,2-dihydro-sn-glycerol-3 and phospholipid), four drug small molecules (ethinylestradiol, artemisinin, The mass spectrum peak of the target analyte can be obtained by both doxorubicin and dexamethasone, as well as the tannic acid molecule. when different amino acid molecules are detected, a quasi-molecular ion peak of a proton can be obtained from the amino acid, but the detection signal-to-noise ratio of each amino acid is different, and by quantum chemical calculation, we find that the amino acid detection effect of the amino acid can be lower, and the signal-to-noise ratio is high. For the detection of the phospholipid, the signal-to-noise ratio of the analyte is high, and the spectrogram is clean and free of matrix interference. We discussed and verified the source of the analyte fragmentation peak. The results of our study further show that Schiff base S1 has certain versatility for acidic small molecules, lipids and alcohols in negative ion mode as MALDI matrix. (3) According to the literature, the compounds 9AA, DMAN and DPN can also be used as the MALDI matrix in the negative ion mode, and the detection of the small molecular analytes has the advantages of less matrix interference. We compared the Schiff base S1 matrix with these basic matrices by the following experiments: 1) the influence of the matrix to the sample peak at different molar ratios; 2) the stability of the substrate under vacuum; and 3) the interference of the matrix peak at different concentrations. 4) solid ultraviolet experiment of the substrate; 5) using quantum chemical means to calculate the effect of each basic matrix on the long-term H-O bond length of the acidic small molecule in the gas phase and the liquid phase and the proton affinity energy (PA) of the alkaline substrates in the gas phase, The proton affinity of the deprotonated proton (PA[M-H]-), the ionization energy (IE) and the electron affinity (EA), and the like. The experimental results show that the Schiff base S1 has the advantages of being stable under vacuum, and being beneficial to quantitative analysis and high sensitivity. In this paper, we report a newly developed Schiff base matrix compound, which can effectively reduce the interference of the matrix to the small molecule analyte in the negative ion mode of MALDI, and realize the simple, rapid and accurate qualitative and quantitative analysis on various compounds such as acid, alcohol and phospholipid. The mechanism of the possible action of the matrix is described in the light of the experimental results and the quantum chemical means.
【学位授予单位】:吉林大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:O657.63
[Abstract]:The matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is characterized by its soft ionization and can be used in the analysis of biological macromolecules such as proteins, nucleic acids and synthetic polymers. At the same time, as the sample preparation is simple, the analysis speed is high, the sensitivity is high, the sample consumption is low, the salt tolerance is good, and the like, and the small molecular sample such as the animal and plant metabolites and the pesticide residue in the actual sample also has an irreplaceable advantage. However, in the actual MALDI-MS detection process, it is often necessary to use the matrix, the traditional matrix produces the matrix ion peak in the small molecule region, the measurement of the small molecular analyte is seriously disturbed, and the reproducibility of the signal intensity of the analyte is poor, which is not conducive to the application in the quantitative analysis of small molecules. Therefore, a new Schiff base compound is developed and used as a MALDI matrix. In the negative ion mode, the detection of small molecular analytes has been successfully achieved. The experimental contents mainly include the following three aspects: (1) a one-step dehydration condensation reaction using aniline and aldehyde substances, Six Schiff base compounds (S1, S2, S3, S4, S5 and S6) were synthesized and used as the MALDI matrix to analyze the fatty acid compounds in the negative ion mode, and the test results of the matrix were evaluated. The reasons for different detection effects of different Schiff bases were discussed using the method of ultraviolet absorption spectrum and quantum chemical calculation. The free fatty acids in the fatty acid standard and the serum are respectively detected by the S1 as the MALDI matrix through the optimization. The fatty acid in serum was extracted by liquid-liquid extraction. The reproducibility of quantitative analysis was improved by adding internal standard (C17: 1) and seed method (improving the uniformity of the co-crystallization of matrix and analyte), and the quantitative analysis of free fatty acids in human serum was achieved. The results show that using the Schiff base S1 matrix to qualitatively and quantitatively analyze the fatty acid has the advantages of no matrix interference, high flux, high sensitivity, simple separation step, no need of derivatization, less sample amount, and the like. (2) The versatility of Schiff base S1 as MALDI matrix in the detection of other acid and alcohol molecules in negative ion mode was investigated. Six kinds of amino acids (tryptophan, lysine, histidine, threonine, leucine, glutamic acid), two kinds of phospholipids (1,2-dihydro-sn-glycerol-3 and phospholipid), four drug small molecules (ethinylestradiol, artemisinin, The mass spectrum peak of the target analyte can be obtained by both doxorubicin and dexamethasone, as well as the tannic acid molecule. when different amino acid molecules are detected, a quasi-molecular ion peak of a proton can be obtained from the amino acid, but the detection signal-to-noise ratio of each amino acid is different, and by quantum chemical calculation, we find that the amino acid detection effect of the amino acid can be lower, and the signal-to-noise ratio is high. For the detection of the phospholipid, the signal-to-noise ratio of the analyte is high, and the spectrogram is clean and free of matrix interference. We discussed and verified the source of the analyte fragmentation peak. The results of our study further show that Schiff base S1 has certain versatility for acidic small molecules, lipids and alcohols in negative ion mode as MALDI matrix. (3) According to the literature, the compounds 9AA, DMAN and DPN can also be used as the MALDI matrix in the negative ion mode, and the detection of the small molecular analytes has the advantages of less matrix interference. We compared the Schiff base S1 matrix with these basic matrices by the following experiments: 1) the influence of the matrix to the sample peak at different molar ratios; 2) the stability of the substrate under vacuum; and 3) the interference of the matrix peak at different concentrations. 4) solid ultraviolet experiment of the substrate; 5) using quantum chemical means to calculate the effect of each basic matrix on the long-term H-O bond length of the acidic small molecule in the gas phase and the liquid phase and the proton affinity energy (PA) of the alkaline substrates in the gas phase, The proton affinity of the deprotonated proton (PA[M-H]-), the ionization energy (IE) and the electron affinity (EA), and the like. The experimental results show that the Schiff base S1 has the advantages of being stable under vacuum, and being beneficial to quantitative analysis and high sensitivity. In this paper, we report a newly developed Schiff base matrix compound, which can effectively reduce the interference of the matrix to the small molecule analyte in the negative ion mode of MALDI, and realize the simple, rapid and accurate qualitative and quantitative analysis on various compounds such as acid, alcohol and phospholipid. The mechanism of the possible action of the matrix is described in the light of the experimental results and the quantum chemical means.
【学位授予单位】:吉林大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:O657.63
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