Proteomics of exhaled breath: methodological nuances and pitfalls.
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.
Kurova
Viktoria S
VS
N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia. kurova@sky.chph.ras.ru
Anaev
Eldar C
EC
Kononikhin
Alexey S
AS
Fedorchenko
Kristina Yu
KY
Popov
Igor A
IA
Kalupov
Timothey L
TL
Bratanov
Dmitriy O
DO
Nikolaev
Eugenie N
EN
Varfolomeev
Sergey D
SD
D000328:Adult; D000388:Air / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D001944:Breath Tests / Q000379:methods; D005260:Female; D006801:Humans; D007633:Keratins / Q000302:isolation & purification; D008297:Male; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012140:Respiratory Tract Diseases / Q000175:diagnosis; D013048:Specimen Handling / Q000379:methods* / Q000592:standards
D015415:Biomarkers; D011506:Proteins; D007633:Keratins