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. 2022 Jun 6;19(1):20.
doi: 10.1186/s12014-022-09348-y.

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

Jörgen Östling  1   2 , Marleen Van Geest  1   3 , Henric K Olsson  4 , Sven-Erik Dahlen  5 , Emilia Viklund  6 , Per M Gustafsson  7 , Ekaterina Mirgorodskaya  8 , Anna-Carin Olin  9
Affiliations

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

Jörgen Östling et al. Clin Proteomics. .

Erratum in

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.

Keywords: Asthma; Biomarker; Breath; Exhaled air; Non-invasive; Non-volatiles; Precision medicine; Proteomics; Small airways.

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Conflict of interest statement

Anna-Carin Olin is reporting competing interests as she is one of the inventors of the PExA method, and boardmember and chairholder of PExA AB. Emilia Viklund is reporting a minor chairhold in PExA AB. Dr Östling reports personal fees from PExA AB during the conduct of the study; and Employed by PExA AB while writing the manuscript but not during the planning and completion of the study.

Figures

Fig. 1
Fig. 1
Distribution of CV for 174 proteins detected in a pooled PEx sample (1 µg PEx/ml), analysed 5 times with the SOMAscan 1.1 K platform. The pooled sample originated from 6 subjects with asthma and 3 healthy volunteers. Proteins were considered detected if RFU values delivered by SomaLogic were larger than LOD in all 5 replicate samples. Limit of detection (LOD) was calculated as 3 times the standard deviation from the mean RFU signal measured from 3 blank samples
Fig. 2
Fig. 2
Assessment of intra-individual variability by visual inspection of Principle Component Analysis (PCA) plot. PEx samples from 3 consecutive PEx samples from 6 asthmatic subjects (red, blue, green, white, black and yellow) were analysed with the SOMAscan 1.1 K platform. Using ANOVA statistical test based variable selection (q < 5.5E−5) 42 out of 114 proteins commonly detected in all 18 samples, were found to discriminate all 6 subjects from each other in a PCA plot, as judged by visual inspection in Omics Explorer software
Fig. 3
Fig. 3
Visualization of results from Gene Ontology (GO) enrichment analysis (GOrilla [28]) matching 207 proteins detected in PEx samples by SOMAscan 1.3 K platform, to the GO Cellular Component sub-domain database. Over represented GO terms are organized in a parent–child based hierarchically structure with color-coded significance levels (Fisher’s exact test), as indicated in the p value colour scale
Fig. 4
Fig. 4
Box plots show examples of SOMAscan data for 6 differentially abundant proteins; a Alpha-1-antitrypsin (SERPINA1), b Interleukin-1 Receptor accessory protein (IL1RAP), c CC motif chemokine 18 (CCL18), d Complement component C1q receptor (CD93), e Immunoglobulin M (IgM), in non-asthma (NA), asthma without (A) and with small airway involvement (A-hLCI), and f Soluble Receptor of Advanced Glycation End products (sRAGE) in never-smokers (NS) and ex-smokers (ExS). Y-axis show normalized abundance (log2 transformation and normalization to mean 0 and variance 1). Box ranges from the 25th to the 75th percentile and median value is marked with dotted line. p values and false discovery rate adjusted p values (q) from various pairwise comparisons are shown over each box plot. Protein abundance data was adjusted for difference in age
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