MASS SPECTROMETRIC ANALYSIS OF EICOSANOIDS AND PROTEINS IN EXHALED BREATH

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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