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 资助的中心拨款提供的资源的众多研究子项目之一，该子项目和研究者 (PI) 可能已从其他 NIH 来源获得主要资助，因此可以在其他 CRISP 机构中得到体现。所列出的是该中心，该中心不一定是研究人员的机构。呼出气冷凝物 (EBC) 含有丰富的挥发性物质、脂质和蛋白质，其中包括类二十烷酸。前列腺素和白三烯以及细胞因子蛋白（例如白细胞介素）以前被认为与肺功能受损有关，EBC 的收集是非侵入性的，并且可以轻松进行，甚至可以由患有肺部疾病的患者进行。气道内壁液体的成分；因此，测量 EBC 中的这些标记物可以提高我们对慢性阻塞性肺病和哮喘等疾病发病机制的了解 (1-2) 从对照受试者和受试者中收集冷凝物。哮喘诊所患者的方法是让每个受试者对冷却至 -20°C 的 R 管进行潮式呼吸 15 分钟C. 采集后，样品保存于-80℃ C，然后使用多种方法制备，包括冻干、一维电泳凝胶分离、溶剂萃取和固相萃取。在这些程序中的一种或多种之后，通过 MALDI-TOF-MS、LC/MS 进行分析。之前对 EBC 中单个类二十烷酸的 ELISA 测量报告表明，这些化合物的含量在 pg/mL 至 ng/mL 范围内 (3)。对 EBC 的各个对照样品进行的蛋白质测定表明总蛋白质的浓度范围为 5 至 20 ¿为了补偿低浓度，将 EBC 样品分成 2-20 组，并使用上述方法浓缩 10-100 倍，在哮喘 EBC 中初步鉴定了几种与炎症和氧化应激相关的类花生酸。临床患者使用微孔 HPLC 色谱柱和在线 ESI-MS 检测血栓素 B2 和 8-异前列腺素的选定离子色谱图，如图 1 和图 1 所示。如下图 2 所示，使用 MALDI-TOF-MS，在一维凝胶、蛋白酶消化和毛细管 LC/ 上获得的 EBC 样品光谱中检测到了 m/z 5000-50,000 范围内的完整蛋白质。对所得肽的 MS/MS 分析有助于鉴定对照受试者 EBC 中的多种蛋白质，我们正在研究这些蛋白质在患者 EBC 中的出现情况和相对丰度。 (1) Montuschi, P. 和 P. Barnes, Trends in Pharmacol.l Sc., 2002, 23, 232。 (2) Hunt, J., .J Allergy and Clin., 2002, 110, 28-34。 (3)Montuschi, P.、M. Corradi、G. Ciabattoni、J. Nightingale、S. Kharitonov 和P. 巴恩斯，J. 护理医学，1999，160，2