【摘要】：基質(zhì)輔助激光解吸離子化質(zhì)譜(MALDI-MS)以其軟電離的特點(diǎn),能夠獲得較完整的分子離子信息,常用于生物大分子如蛋白、核酸和合成聚合物的分析。同時(shí),由于樣品準(zhǔn)備簡單,分析速度快,靈敏度高,樣品消耗少,耐鹽性好等諸多優(yōu)點(diǎn),在分析實(shí)際樣品中動(dòng)植物代謝物和農(nóng)藥殘留等小分子樣品也有著不可替代的優(yōu)勢。然而,在實(shí)際MALDI-MS檢測過程中經(jīng)常需要使用基質(zhì),傳統(tǒng)基質(zhì)在小分子區(qū)域產(chǎn)生基質(zhì)離子峰,嚴(yán)重干擾小分子分析物的測定,而且分析物信號(hào)強(qiáng)度的重現(xiàn)性較差,不利于其在小分子定量分析中的應(yīng)用。因此,本文開發(fā)了一種新的Schiff堿類化合物,并將其用作MALDI基質(zhì),在負(fù)離子模式下,成功實(shí)現(xiàn)了對(duì)小分子分析物的檢測,實(shí)驗(yàn)內(nèi)容主要包含以下三個(gè)方面:(1)使用苯胺和醛類物質(zhì)一步脫水縮合反應(yīng),合成了六種Schiff堿類化合物(S1、S2、S3、S4、S5和S6),將其用作MALDI基質(zhì)在負(fù)離子模式下分析了脂肪酸類化合物,評(píng)價(jià)了基質(zhì)的測試效果。使用紫外吸收光譜和量子化學(xué)計(jì)算的方法探討了不同Schiff堿基質(zhì)產(chǎn)生不同檢測效果的原因。經(jīng)過優(yōu)化,最終選取S1作為MALDI基質(zhì)分別檢測了脂肪酸標(biāo)準(zhǔn)品和血清中的游離脂肪酸。使用液液萃取的方法對(duì)血清中脂肪酸進(jìn)行粗提取。采取加入內(nèi)標(biāo)(C17:1)和種子法點(diǎn)樣(提高基質(zhì)與分析物共結(jié)晶的均勻度)兩種方法來提高定量分析的重現(xiàn)性,最終實(shí)現(xiàn)了人血清中游離脂肪酸的定量分析。結(jié)果顯示:使用Schiff堿S1基質(zhì)定性定量分析脂肪酸具有無基質(zhì)干擾、高通量、高靈敏度、分離步驟簡單、無需衍生化、需樣量少等優(yōu)點(diǎn)。(2)考察了Schiff堿S1作為MALDI基質(zhì)在負(fù)離子模式下檢測其它酸性及醇類小分子的通用性。使用S1基質(zhì)檢測了六種氨基酸(色氨酸、賴氨酸、組氨酸、蘇氨酸、亮氨酸、谷氨酸)、兩種磷脂(1,2-二油酰-sn-甘油-3磷脂和鞘磷脂)、四種藥物小分子(炔雌醇、青蒿素、阿霉素和地塞米松)以及單寧酸分子,均可獲得目標(biāo)分析物的質(zhì)譜峰。檢測不同氨基酸分子時(shí),均能得到氨基酸失去一個(gè)質(zhì)子的準(zhǔn)分子離子峰,但各氨基酸的檢測信噪比有所不同,通過量子化學(xué)計(jì)算,我們發(fā)現(xiàn)電離能較低的氨基酸檢測效果較好,信噪比高。對(duì)于磷脂的檢測,分析物信噪比高,譜圖干凈無基質(zhì)干擾。我們對(duì)分析物碎裂峰的來源進(jìn)行了機(jī)理討論及驗(yàn)證。我們的結(jié)果進(jìn)一步顯示:Schiff堿S1作為MALDI基質(zhì)在負(fù)離子模式下,對(duì)于酸性小分子、脂類及醇類物質(zhì)具有一定的通用性。(3)據(jù)文獻(xiàn)報(bào)道,化合物9AA、DMAN和DPN也可作為MALDI基質(zhì)在負(fù)離子模式下使用,檢測小分子分析物具有基質(zhì)干擾少的優(yōu)點(diǎn)。我們通過以下幾個(gè)實(shí)驗(yàn)將Schiff堿S1基質(zhì)與這些堿性基質(zhì)進(jìn)行了比較:1)基質(zhì)與樣品不同摩爾比下對(duì)樣品峰強(qiáng)的影響;2)基質(zhì)真空下的穩(wěn)定性;3)在不同濃度下檢測分析物時(shí)基質(zhì)峰的干擾;4)基質(zhì)的固體紫外實(shí)驗(yàn);5)使用量子化學(xué)手段,計(jì)算各堿性基質(zhì)在氣相與液相中分別對(duì)酸性小分子的羧基H-O鍵長的影響以及這些堿性基質(zhì)在氣相中的質(zhì)子親和能(PA)、去質(zhì)子的質(zhì)子親和能(PA[M-H]-)、電離能(IE)和電子親和能(EA)等。實(shí)驗(yàn)結(jié)果顯示:Schiff堿S1作為基質(zhì)具有真空下穩(wěn)定、利于定量分析、靈敏度高等優(yōu)勢。在這篇論文中,我們報(bào)道了一種新開發(fā)的Schiff堿類基質(zhì)化合物,在MALDI負(fù)離子模式下可有效減少基質(zhì)對(duì)小分子分析物的干擾,對(duì)酸性,醇類和磷脂等多種化合物實(shí)現(xiàn)簡單、快速、準(zhǔn)確地定性定量分析。我們結(jié)合實(shí)驗(yàn)結(jié)果和量子化學(xué)手段闡述了基質(zhì)可能的作用機(jī)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.63

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本文編號(hào)：2314122