ИСТИНА

A methodological approach to increase protein identifications in exhaled breath condensate Kozyr A.S.1, Indeykina M.I.1, Zakharova N.V.1, Ryabokon A.M. 1, 4, Kononikhin A.S.1,2,3, Nikolaev E.N. 3 1 Institute of Biochemical Physics named after N.M. Emanuel of the Russian Academy of Sciences, Moscow 2 V.L. Talrose Institute for Energy Problems of Chemical Physics of the Russian Academy of Sciences, Moscow 3 Skolkovo Institute of Science and Technology, Skolkovo 4 Lomonosov Moscow State University E–mail: anykozyr@yandex.ru Introduction: Exhaled breath condensate (EBC) is a promising target for the detection of biomarkers of socially significant lung pathologies, including lung cancer, as it contains thousands of compounds that reflect lung metabolism. Protein and peptide EBC biomarkers are of particular interest, and require the high-sensitive methods for analysis, such as high-resolution mass-spectrometry. Currently, the lack of standardized EBC collection and sample preparation protocols still remains a major obstacle to widespread use of EBC analysis in the clinic. Herein, in order to reveal the conditions for the high-yield identification of EBC proteins, a particular sampling modification was proposed. Methods: EBC samples from healthy controls and lung cancer patients were collected with RTube® device (Respiratory Research, Inc., Charlottesville, VA). After EBC remove, the collecting tube was additionally rinsed with 1 ml of methanol. EBC and methanol fractions were further estimated by poly-acrylamide gel electrophoresis (PAGE), and peptides obtained after tryptic digestion were analyzed by LC-ESI-MS/MS using a nano-LC Agilent 1100 system coupled to 7T LTQFT Ultra tandem high resolution mass-spectrometer (the equipment of the Center for Science and Technology at the IBCF of the Russian Academy of Sciences “New materials and new technologies”) in DDA mode followed by detailed data analysis. Results: Both PAGE and MS studies confirmed that methanol fraction was more enriched with proteins (~1.5 fold) and peptides (~2 fold) than EBC. Thus, the additional rinsing with methanol essentially reduces the protein sorption at the inner surface of the collecting device. The analysis of both EBC and methanol fraction provided the essential improvement in EBC peptide and protein identifications. Conclusions: The proposed above methodological improvement is extremely relevant for creation of reliable methods for EBC proteome and peptidome analysis, which may provide highly important tool for timely noninvasive diagnostics of a number of socially significant diseases, including lung cancer. The research was supported by RFBR grant # 18-29-09158 MK.