Background: The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Methods: Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. Results: EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Conclusions: Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations. Clin Chem Lab Med 2009;47:706-12.
Analysis of samples of exhaled breath condensate (EBC) is a promising noninvasive method for the control of the status of the human respiratory system. In the present work, the proteome of the EBC samples received from a patient with lung dystrophy at different times before and after bilateral lung transplantation is analyzed by ion cyclotron resonance mass spectrometry. Qualitative protein composition of EBC samples obtained during the first month correlates with the clinical data on the acceptance of the transplanted lungs (allograft). Fifteen months after the lung surgery, the protein spectrum was similar to the normal composition of EBC proteins. This result agrees with the medical conclusion about normal lung functioning. The results suggest that the mass spectrometric monitoring of the protein spectra of EBC may be a tool for noninvasive pulmonological diagnostics.Key words: ion cyclotron resonance mass spectrometry, proteomic analysis, exhaled breath condensate, diagnostics of respiratory diseases.Recent instrumental advances in mass spectrometry (MS) and chromatography allow perfection of the meth ods of medical diagnostics based on the studies of the structure and function of biomacromolecules. The sensi tivity of the most recent mass spectrometers allows for the detection of femtomole concentration of proteins and pep tides with the dynamic range of concentrations of several orders of magnitude. The accuracy of mass determination of charged species with the state of the art methods reach es one millionth part of the ion mass. These characteristics are pertinent, in particular, to the Fourier transform ion cyclotron resonance mass spectrometers (FT ICR MS). Coupling of high performance liquid chromatography with FT ICR mass spectrometry ensures the high performance of the analysis and allows the quantitative analysis of the components of complex mixtures.In the present study, the potential of FT ICR chro mato mass spectrometry was employed for identification of the protein composition of the exhaled breath conden sate (EBC) of a person who had undergone lung surgery. The analysis of EBC is a promising method for the nonin vasive diagnosis of human respiratory system, but the prep aration of samples for the analysis significantly differs from that used for blood or urine. The method of EBC collec tion and analysis of protein composition by chromato mass spectrometry has previously been practized with ma terial obtained from healthy non smoking donors. Below we present the results of the qualitative analysis of EBC protein composition of a patient with bullous emphysema who underwent bilateral lung transplantation. Thorough medical monitoring of the patient in pre and post sur gery periods allowed us to obtain a unique set of samples of exhaled breath condensate.
Polyurethane foam (PUF) has been suggested as a solid polymeric reagent for determination of nitrite. The determination is based on the diazotization of end toluidine groups of PUF with nitrite in acidic medium followed by coupling of polymeric diazonium cation with 3-hydroxy-7,8-benzo-1,2,3,4-tetrahydroquinoline. The intensely colored polymeric azodye formed in this reaction can be used as a convenient analytic form for the determination of nitrite by diffuse reflectance spectroscopy (c (min) = 0.7 ng mL(-1)). The possibility of using a desktop scanner, digital camera, and computer data processing for the numerical evaluation of the color intensity of the polymeric azodye has been investigated. A scanner and digital camera can be used for determination of nitrite with the same sensitivity and reproducibility as with diffuse reflectance spectroscopy. The approach developed was applied for determination of nitrite in river water and human exhaled breath condensate.
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