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In the clinical setting, it is common practice to collect blood and urine from patients for a myriad of tests but recent years have seen a move towards less invasive methods of sampling, where possible. Not all patients are comfortable with these classical methods and there are some who are downright afraid, like those with needle phobias. For some medical conditions, blood or urine might be the only suitable fluids to analyse but others offer more flexibility. In the search for protein biomarkers of respiratory diseases such as asthma and cystic fibrosis, bronchoalveolar lavage fluid and sputum have been used as alternatives but another possibility has been explored by a group of scientists in Belgium. They reasoned that this type of disease would alter the protein composition on the breath. Collection of exhaled breath condensate (EBC) would be a better non-invasive procedure, since patients can simply sit and breathe normally while sampling takes place. There have been a few studies of EBC but the protein profile necessary for detailed proteome analysis and comparison has not yet been established. The main problem with EBC in the proteomics setting is the low concentration of proteins, about 1 µg/mL. So the scientists developed an EBC collection and treatment method that optimises protein levels and reduces losses. Karolien Bloemen and colleagues from the Flemish Institute for Technological Research in Mol, the University Hospital of Antwerp and the University of Antwerp tested their procedures on EBC from 6 healthy smokers and 6 healthy non-smokers. The subjects were asked to breathe into a cooled tube for 15 minutes, inhaling and exhaling normally by a process referred to as tidal breathing. The breath condensed and collected in the tubes then was transferred to a smaller container and frozen until analysis. Following protein concentration, the proteins were digested with trypsin and the peptides mixtures formed were fractionated by HPLC to give 60 fractions per sample, one every 30 seconds, that were collected on a target for MALDI mass spectrometry analysis. HPLC was preferred to 2D gel electrophoresis because it gave better results with a run-to-run reproducibility coefficient of variation of 1.2%. The peptide mass spectra were determined in scan mode and by tandem mass spectrometry. Proteins were identified by searching the SwissProt sequence database. Three types of breath collection tube were compared using standard solutions of bovine serum albumin with commercial Protein LoBind tubes giving high recoveries of 95%. Two other containers, glass vials and plastic tubes, allowed too much protein to be lost resulting in recoveries of 75 and 55%, respectively. The protein concentration method was also optimised. Two types of ultramembrane centrifugation devices gave recoveries of just 25%. Freeze drying was highly irreproducible with recoveries ranging from 25 to 80%. Vacuum concentration fared better but the researchers questioned the use of protein solubilisation over several hours, which might induce instability. The most reliable method used precipitation with the dye pyrogallol red and gave recoveries of 80% but the dye could not subsequently be removed. So the team selected precipitation on Poros 20 R2 beads, with consistent recoveries of 50%. In the final optimised procedure, 1 mL of EBC was transferred from the collection tube to the smaller container and the beads were added to bind the proteins. The aqueous phase was removed after centrifugation and the proteins were on-bead digested with trypsin in a LoBind tube. Seven proteins were identified in the EBC from smokers and non-smokers but they were all more abundant in the smokers. The most abundant proteins were four cytokeratins, the increase in smokers being consistent with airway epithelial damage. The remaining three proteins were identified for the first time in EBC due to the improved isolation procedures. They were calgranulin A, haemoglobin and lysozyme C. An eighth protein, calgranulin B, was identified solely in EBC from smokers. It is associated with acute and chronic inflammation and has been reported at increased levels in bronchoalveolar lavage fluid from smokers. Calgranulin B is about 20 times less abundant than the most abundant cytokeratin, confirming the sensitivity of the sampling method. The researchers accept that this is a limited pilot study, collecting and analysing just 12 samples in total, but they argue that the results show the potential of the method for collecting and comparing low abundance proteins in EBC. The sensitivity will be improved when the EBC collection device is modified, facilitating the identification of more low abundant proteins. Currently about 80% of the proteins are lost on the internal surface. Once accepted, the collection system will provide an easy, non-invasive way of collecting EBC and will be particularly suitable for children or patients with chronic airway complaints. There is no need for sustained blowing or forced respiratory manoeuvres as the natural process of tidal breathing is sufficient. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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