A novel non-invasive method allowing for discovery of pathologically relevant proteins from small airways

Abstract

Background There is a lack of early and precise biomarkers for personalized respiratory medicine. Breath contains an aerosol of droplet particles, which are formed from the epithelial lining fluid when the small airways close and re-open during inhalation succeeding a full expiration. These particles can be collected by impaction using the PExA® method (Particles in Exhaled Air), and are derived from an area of high clinical interest previously difficult to access, making them a potential source of biomarkers reflecting pathological processes in the small airways. Research question Our aim was to investigate if PExA method is useful for discovery of biomarkers that reflect pathology of small airways. Methods and analysis Ten healthy controls and 20 subjects with asthma, of whom 10 with small airway involvement as indicated by a high lung clearance index (LCI ≥ 2.9 z-score), were examined in a cross-sectional design, using the PExA instrument. The samples were analysed with the SOMAscan proteomics platform (SomaLogic Inc.). Results Two hundred-seven proteins were detected in up to 80% of the samples. Nine proteins showed differential abundance in subjects with asthma and high LCI as compared to healthy controls. Two of these were less abundant (ALDOA4, C4), and seven more abundant (FIGF, SERPINA1, CD93, CCL18, F10, IgM, IL1RAP). sRAGE levels were lower in ex-smokers (n = 14) than in never smokers (n = 16). Gene Ontology (GO) annotation database analyses revealed that the PEx proteome is enriched in extracellular proteins associated with extracellular exosome-vesicles and innate immunity. Conclusion The applied analytical method was reproducible and allowed identification of pathologically interesting proteins in PEx samples from asthmatic subjects with high LCI. The results suggest that PEx based proteomics is a novel and promising approach to study respiratory diseases with small airway involvement.

Activation of the Complement and Coagulation Systems in the Small Airways in Asthma

“…Results from the present study focussing on differences between subjects with asthma with and without small airway dysfunction and healthy controls (HC), respectively, have been presented earlier [20]. During the analyses of protein profiles in exhaled particles, it has been evident that proteins belonging to the complement and coagulation systems are overrepresented and seem of particular interest, both in the present study and also in another study population including smokers [21].…”

“…SOMAscan (SomaLogic Inc, Boulder, USA) is an aptamer-based proteomics platform that uses slow off-rate-modified DNA aptamers (SOMAmers) as high-affinity protein capture reagents to quantify proteins. PEx sample preparation and SOMAscan analysis has been previously described in detail [20, 21]. Prior to SOMAscan analysis, the volume of sample buffer was adjusted to reach the same concentration of PEx in all samples in order to normalize the samples for the differences in the collected amount of PEx.…”

“…Subjects were recruited from our previous studies or by an advertisement in a daily paper. Some results from the study have been presented earlier but with a different general approach [20].…”

Particles in Exhaled Air (PExA): Clinical Uses and Future Implications

“…In addition to particle flow rate, samples can be analysed through mass spectrometry for lipidomics or enzyme-linked immunosorbent assay (ELISA) for proteomics to establish the chemical composition of the small airways, allowing differences to be established between physiology and pathology [19]. Particles sampled and assessed through PExA have considerable potential; Bredberg et al (2012) identified 120 different proteins that can be detected, with up to 80% being concurrently detected via invasive bronchoscopy, and Östling et al identified 207 proteins from PExA samples, with phenotypic distributions depending on the smoking status [37]. An additional advantage of using PExA (in comparison to both BAL and tracheal aspirate) is that it does not involve administering foreign substances into the respiratory system that can alter or dilute the respiratory fluid composition [13].…”

“…and Östling et al identified 207 proteins from PExA samples, with phenotypic distributions depending on the smoking status [37]. An additional advantage of using PExA (in comparison to both BAL and tracheal aspirate) is that it does not involve administering foreign substances into the respiratory system that can alter or dilute the respiratory fluid composition [13].…”