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题名	基质辅助激光解吸电离飞行时间质谱的设计与应用研究
作者	栗越妍
学位类别	博士
答辩日期	2014
授予单位	中国科学院研究生院
授予地点	北京
导师	束继年
关键词	MALDI Fungal spore lipid β-Sitosterl Oxidation ，基质辅助激光解吸电离 真菌孢子 脂质 β-谷甾醇 氧化
其他题名	Matrix-assistant laser desorption/ionization time-of-flight mass spectrometer:development and application
中文摘要	生物气溶胶在大气环境中非常普遍。它们不仅对人体健康有直接的影响；还会通过影响全球气候变化、大气化学等过程，对人体健康产生间接的影响。本论文着眼于基质辅助激光解吸电离飞行时间质谱（matrix-assistant laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS）的设计研发及其在生物气溶胶脂质生物标记物检测方面的应用研究。通过MALDI仪器调试，确定仪器运行参数。通过MALDI实验条件优化，获取真菌担孢子脂质生物信息；并进一步分析了植物甾醇定性和定量的MALDI质谱图信息。 通过MALDI仪器调试运行，该自研MALDI-TOF MS可以检测质量大于200 Da的分子，分辨率达到2000（m=1000 Da, m/△m），灵敏度4 pmol，在1000 Da检测范围内质量准确度可达7 ppm。分析大豆卵磷脂混合物，在正离子检测模式下其MALDI-TOF质谱图以磷脂酰胆碱为主；负离子检测模式下的质谱图则以磷脂酰肌醇和磷脂酰乙醇胺为主。大豆甲醇脂质粗提物的MALDI-TOF质谱图主要以甘油二酯和磷脂酰胆碱为主。 通过MALDI实验条件优化，考察不同实验因素（基质种类、溶剂、激光能量、阳离子化试剂、分析物浓度）对质谱脂质检测方法的影响。配合简单的脂质提取过程，六种担孢子（黑木耳(A. auricula)、双孢蘑菇(A. bisporus)、斑玉蕈(H. marmoreus)、香菇(L. edodes)、侧耳(P. ostreatus)和草菇(V. volvacea)）的脂质提取液与基质溶液（2,4-DHB/甲醇，10 mg mL-1）等体积混合，在此实验条件下获得的担孢子质谱图包含最丰富的脂质生物信息。极性甘油磷脂（比如甘油磷脂酸、磷脂酰胆碱类）是测试孢子样品中最广泛被检测到的脂质分子。其他甘油磷脂，包括磷脂酰乙醇胺、磷脂酰丝氨酸和磷脂酰甘油类脂质在大部分的担孢子样品中也可以检测到。中性脂质只在部分测试孢子样品中有发现，像A. auricula、A. bisporus和P. ostreatus孢子样品中可以检测到甘油二酯分子，而甘油三酯分子只在H. marmoreus孢子样品中被检测到。在此研究基础上，进一步考察了甾醇类脂质——β-谷甾醇的MALDI分析方法。最佳的MALDI-TOF检测方法：分析物与基质溶液（2,4-DHB/异丙醇:水（9:1），10 mg mL-1）等体积混合，激光能量13.5 mJ cm-2。β-谷甾醇MALDI质谱图的特征性质谱峰由两部分组成：第一部分是β-谷甾醇分子的特征性质谱峰，包括m/z 396.6，397.6，436.9和452.6，分别对应[M-H2O]+，[M+H-H2O]+，[M+Na]+和[M+K]+型离子；第二部分是β-谷甾醇一元氧化分子的特征性质谱峰，包括m/z 412.7，413.7和 431.9，分别对应[M′-H2O]+，[M′+H-H2O]+和[M′+H]+型离子。MALDI质谱图的量化分析得到β-谷甾醇回归方程y=0.004x+3.198，R2=0.992。线性检测范围在12.1到1207.5 μM之间。浓度检测下限为10 nM。 本论文通过MALDI-TOF MS的设计研发及应用研究表明：MALDI-TOF MS提供可靠且具有重复性的脂质检测方法。配合简单的脂质提取过程获取的脂质MALDI质谱图具有种-属专属性，可以用来区分真菌孢子种类；MALDI-TOF MS可以应用于定性及定量检测植物甾醇类脂质，这一检测方法可以为营养学、食品科学、医学或环境科学提供快速便捷准确的分析方法 。
英文摘要	Bioaerosols are universal in the atmosphere. Bioaerosols effect human health directly; they also have impact on atmospheric climate or chemistry process, which effect human health indirectly. The paper focused on the development and application of matrix-assistant laser desorption/ionization time-of-flight mass spectrometer. Various parameters were optimized to assure optimal running state of mass spectrometer, to obtain lipid information of fungal spore, and to analyze qualitative and quantitative profile of β-sitosterol. The laboratory-built MALDI-TOF MS can detect molecular mass greater than 200 Da. The mass resolution of the instrument reaches 2000 (m=1000, m/Δm). The detection limit is about 4 pmol. The mass accuracy is 7 ppm in the range of m/z 1000. Soybeans were chosen as a source of phospholipid (PL) classes that can produce negative ions and also contain significant amounts of phosphatidyl cholines (PCs) Positive spectrum was obtained but only peaks related to the PCs were detected. A negative-ion spectrum for this mixture included representative peaks related to phosphatidyl inositols and phosphatidyl ethanolamines (PEs). The crude methanolic extract of soybean was analyzed in the positive mode. Peaks corresponding to all expected diacylglycerol (DG) and phosphatidyl choline (PC) classes could be detected. The matrix compound, solvent system, laser intensity, alkali ion, and analyte concentration for sample preparation were optimized to acquire reproducible and informative mass spectra of the analyte. Lipids were extracted from basidiospores of Auricularia auricula, Agaricus bisporus, Hypsizygus marmoreus, Lentinus edodes, Pleurotus ostreatus, and Volvariella volvacea. Lipids were observed by mixing the lipid extracts with equal volumes of 2,4-DHB/methanol solution (10 mg mL-1). Polar phospholipids (PLs) (e.g. phosphatidic acids and PCs) were the major lipid components observed in all sampled basidiospores. Other membrane-associated PLs, including PEs, phosphatidyl serines, and phosphatidyl glycerols were detected in most of the sampled fungal spores. Neutral lipids were also observed. Diacylglycerols were detected in A. auricula, A. bisporus, and P. ostreatus spores, whereas triacylglycerol was detected only in H. marmoreus spores. Further more the best MALDI spectrum of β-sitosterol was obtained by mixing the analyte solution with an equal volume of 2,4-DHB/iPrOH:H2O (9:1) solution (10 mg mL-1) at 13.5 mJ cm-2 laser intensity. Under the optimized experimental conditions, β-sitosterol generated intense peaks consisting of the initial lipids and their oxidation products. The former included mass peaks at m/z 396.6, 397.6, 436.9, and 452.6, which corresponded to the [M-H2O]+, [M+H-H2O]+, [M+Na]+, and [M+K]+ ions of β-sitosterol, respectively. The latter included mass peaks at m/z 412.7, 413.7, and 431.9, which corresponded to the [M′-H2O]+, [M′+H-H2O]+, and [M′+H]+ ions of mono-oxidized β-sitosterol, respectively. A quantitative analysis of β-sitosterol was also conducted using a matrix peak. Calibration curves obtained for β-sitosterol showed a good linear range of three orders of magnitude with a regression coefficient square of 0.992. The linear ranges were between 12.1 and 1207.5 μM. The detection limit was 10 nM. The researches on development and application of MALDI-TOF MS reveal that MALDI-TOF MS can be used to obtain lipid profiles with reliable speed and reproducibility. Lipids were extracted from fungal spores with a simple extraction procedure. Species-specific lipid profiles of basidiospores can be used to differentiate fungal spores. The results also implicate that the MALDI-TOF MS can offer identification and quantification information of phytosterol molecules. The reported method may provide a potential tool for a rapid and reliable analysis of nutrition, food science, medicine, and environmental science.
内容类型	学位论文
源URL	[http://ir.rcees.ac.cn/handle/311016/35200]
专题	生态环境研究中心_大气环境科学实验室
推荐引用方式 GB/T 7714	栗越妍. 基质辅助激光解吸电离飞行时间质谱的设计与应用研究[D]. 北京. 中国科学院研究生院. 2014.

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