Early diagnosis of cancer provides the greatest chance of a cure or favourable Analysis of exhaled breath condensate (EBC) has recently been proposed as a non-invasive method to diagnose early-stage lung cancer. Since this sample type has not been comprehensively profiled before; here, we investigated variables that may affect protein yield in EBC, prior to constructing protein profiles of human breath from a lung cancer patient and a healthy control.
Using EBC from healthy volunteers, we assessed the effect on total EBC protein of storage tube, method of sample concentration, cooling temperature, use of protease inhibitors and filters/nose-clips and the effect of delaying processing for up to 24hrs. Total protein was quantitated using the Protein Assay Kit on a Qubit 3.0 Fluorometer. Finally, protein mapping of EBC from a patient with NSCLC adenocarcinoma and from an age- and gender-matched control volunteer was performed by Information Dependent Acquisition MS on a Sciex 6600 TripleTOF.
We determined that EBC should be collected at lower cooling temperatures (-80°C) for greatest EBC volume and protein yield. EBC should be stored in plastic with the addition of protease inhibitors to ensure stability during sample processing as well as long-term stability of the sample (assessed up to 2 years post collection). In a preliminary MS profile, 57 proteins were identified, including keratins, mucins and inflammatory proteins. Differentially expressed proteins (n=21, FC>1.5) between lung cancer and control EBC include several serpins (A3/B4/B7/B12), S100 proteins (A7/A11/A14), as well as proteases and lipocalins known for their involvement in lung cancer development and progression.
Understanding the factors that affect EBC yield and quality is crucial for the generation of reproducible and accurate EBC proteomic profiles. Improving our knowledge how expression of these lung proteins change during carcinogenesis is crucial to provide an “exhaled biomarker fingerprint” of lung cancer.