Micro-scale TMT Proteomics for Biomarker Discovery from Exhaled Breath Condensate in a Longitudinal Study

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Exhaled breath condensate proteomics represent a low-cost, non-invasive alternative for examining upper respiratory health. EBC has previously been used for the discovery and validation of detected exhaled volatiles and non-volatile biomarkers of disease related to upper respiratory system distress such as asthma, COPD, lung cancer, and secondary respiratory infections. Emergent utilization of EBC for functional proteomic analysis provides an unbiased snapshot of the biological composition of the upper airway in real time. Characterizing the biological landscape of the EBC proteome proves challenging due to sample collection variability and low detection sensitivity. In this study we overcome some of the technical challenges by applying recently developed microscale proteomic techniques for developing a novel isobaric labeling technique using tandem mass tags (TMT) to enable reliable, relative quantification of low abundance peptides/proteins in breath with significantly improved biomarker detection across a longitudinal cohort of healthy volunteer. With this approach, we achieve deeper proteomic coverage, and report a healthy baseline proteome for both qualitative and quantitative TMT coverage in EBC.  Here, we report a total of 235 quantifiable proteins corresponding to 1,877 non-redundant peptides for evaluating sample collection reproducibility and establishing a healthy (human host) baseline EBC biomarker proteome studies. This work provides a path for further in-depth investigations of perturbed EBC protein expression profiles and showcases the value of using non-invasive collection method techniques for clinically relevant biomarker discovery.