MASS SPECTROMETRIC ANALYSIS OF EICOSANOIDS AND PROTEINS IN EXHALED BREATH

呼出气中类花生酸和蛋白质的质谱分析

• 批准号: 7369265
• 负责人: GEORGE T O'CONNOR
• 金额: $ 2.39万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369265
• 关键词: MASS; SPECTROMETRIC; ANALYSIS; EICOSANOIDS; PROTEINS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Exhaled breath condensate (EBC) harbors a rich mixture of volatile species, lipids, and proteins. Among these are the eicosanoids, such as prostaglandins and leukotrienes, and cytokine proteins, such as the interleukins, which have previously been associated with impaired pulmonary function. Collection of EBC is non-invasive and can be performed easily, even by patients with pulmonary disease. It has been suggested that EBC reflects the composition of the airway lining fluid; therefore, measurement of these markers in EBC may improve our understanding of the pathogenesis of diseases such as chronic obstructive pulmonary disease and asthma. (1-2) Condensate was collected from both control subjects and asthma clinic patients by having each subject breathe tidally for 15 minutes into an R-tube that was cooled to -20 ¿C. After collection, the samples were stored at -80 ¿C, and then were prepared using several methods, including lyophilization, separation on 1D electrophoretic gels, solvent extraction, and solid-phase extraction. After one or more of these procedures, samples were analyzed by MALDI-TOF-MS, LC/MS, and capillary LC/MS/MS. Previous reports on ELISA measurements of individual eicosanoids in EBC indicate that these compounds are present at levels in the range of pg/mL to ng/mL (3). Our total protein assays performed on individual control samples of EBC indicated the concentration of total protein ranged from 5 to 20 ¿g/mL. To compensate for the low concentrations, EBC samples were pooled in groups of 2-20 and concentrated 10-100-fold using the methods described above. Several eicosanoids associated with inflammation and oxidative stress were tentatively identified in the EBC of asthma clinic patients using a microbore HPLC column and online ESI-MS. The selected ion chromatograms for thromboxane B2 and 8-isoprostane are shown in Figures 1 and 2 below. With MALDI-TOF-MS, intact proteins were detected over the range from m/z 5000-50,000 in the spectra obtained for samples of EBC obtained from control subjects. Separation on 1-D gels, protease digestion and capillary LC/MS/MS analyses of the resulting peptides has facilitated the identification of multiple proteins in EBC of control subjects. We are investigating the occurrence and relative abundance of these proteins in the EBC of patients. (1)Montuschi, P. and P. Barnes, Trends in Pharmacol.l Sc., 2002, 23, 232. (2) Hunt, J., .J Allergy and Clin. Immunol., 2002, 110, 28-34. (3)Montuschi, P., M. Corradi, G. Ciabattoni, J. Nightingale, S. Kharitonov, and P. Barnes, Am. J. Respir. Crit. Care Med., 1999, 160, 2

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。呼出气冷凝物（EBC）含有丰富的挥发性物质、脂质和蛋白质的混合物。其中包括类花生酸，如白藜芦醇和白三烯，以及细胞因子蛋白，如白细胞介素，它们以前与肺功能受损有关。EBC的收集是非侵入性的，可以很容易地进行，即使是患有肺部疾病的患者。有人认为，EBC反映了气道衬里液的组成，因此，EBC中这些标志物的测量可能会提高我们对慢性阻塞性肺疾病和哮喘等疾病的发病机制的理解。(1-2)通过让每个受试者潮式呼吸15分钟进入冷却至-20 ℃的R管，从对照受试者和哮喘门诊患者中收集冷凝物。采集后，将样品储存在-80 ℃下，然后使用几种方法制备，包括冻干、一维电泳凝胶分离、溶剂萃取和固相萃取。在这些程序中的一个或多个之后，通过MALDI-TOF-MS、LC/MS和毛细管LC/MS/MS分析样品。先前关于EBC中单个类二十烷酸的ELISA测量的报告表明，这些化合物以pg/mL至ng/mL范围内的水平存在（3）。我们对EBC的单个对照样品进行的总蛋白测定表明，总蛋白的浓度范围为5至20 μ g/mL。为了补偿低浓度，将EBC样品以2 - 20份为一组合并，并使用上述方法浓缩10 - 100倍。使用微孔HPLC柱和在线ESI-MS，在哮喘临床患者的EBC中初步鉴定了几种与炎症和氧化应激相关的类花生酸。血栓烷B2和8-异前列烷的选定离子色谱图见下图1和2。使用MALDI-TOF-MS，在从对照受试者获得的EBC样品的光谱中，在m/z 5000 - 50，000的范围内检测到完整蛋白质。1-D凝胶分离，蛋白酶消化和毛细管LC/MS/MS分析所得到的肽促进了多种蛋白质的EBC的控制对象的识别。我们正在研究这些蛋白质在患者EBC中的发生率和相对丰度。 （1）Montuschi，P.和P.巴恩斯，药理学趋势，2002，23，232。(2)亨特，J.，. J Allergy and Clin. Immunol.，2002，110，28 - 34。（3）Montuschi，P.，M.科拉迪湾Ciabattoni，J. Nightingale，S. Kharitonov，and P.巴恩斯，Am. J. Respir. Crit. Care Med.，1999年，160，2