ИСТИНА

Exhaled breath condensate (EBC) reflects the composition of the airway-lining fluid and may contain biomarkers of diseases of respiratory system. The aim of this study is to identify proteins in EBC of patients with chronic obstructive pulmonary disease (COPD) and pneumonia using two techniques of proteome identification. Seventeen COPD and thirteen pneumonia samples were collected using the Jaeger ECoScreen (VIASYS Healthcare, Germany), freeze dried, treated with trypsin and analyzed by nanoflow LC-MS/MS with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Six COPD samples were mixed and applied to 2D-PAAGE by Mini-PROTEAN system (Bio-RAD, USA). Silver stained spots were analyzed by MALDI-TOF-MS using a Microflex device (Bruker Daltonics, Germany). Using 2D-PAAGE and MALDI-TOF-MS we were able to show that EBC samples from patients with COPD contain whole “normal” keratins that were detected also in EBC of healthy donors. Using LC-MS/MS, a method of comparable sensitivity, specific peptides of “abnormal” keratins 3, 4, 8 were identified in COPD samples. Keratin set identified in samples from patients with acute pneumonia was more varied. Peptides of Plakoglobin, Desmoplakin, Alpha-1-acid glycoprotein, Filaggrin, Dynein, Collagen, Hornerin were discovered in COPD and pneumonia EBC samples. They are uncharacteristic of healthy EBC samples. None of these proteins was identified as a whole in 2D-PAA gels. These peptides seem to appear in airway-lining fluid due to proteolysis in respiratory tract tissue. In conclusion, each of “abnormal” peptides, as well as their combinations, may have diagnostic value.