Identification of Proteomic Signatures in Chronic Obstructive Pulmonary Disease Emphysematous Phenotype

Abstract

Chronic obstructive pulmonary disease (COPD) is a highly heterogeneous disease. Emphysematous phenotype is the most common and critical phenotype, which is characterized by progressive lung destruction and poor prognosis. However, the underlying mechanism of this structural damage has not been completely elucidated. A total of 12 patients with COPD emphysematous phenotype (COPD-E) and nine patients with COPD non-emphysematous phenotype (COPD-NE) were enrolled to determine differences in differential abundant protein (DAP) expression between both groups. Quantitative tandem mass tag-based proteomics was performed on lung tissue samples of all patients. A total of 29 and 15 lung tissue samples from patients in COPD-E and COPD-NE groups, respectively, were used as the validation cohort to verify the proteomic analysis results using western blotting. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted for DAPs. A total of 4,343 proteins were identified, of which 25 were upregulated and 11 were downregulated in the COPD-E group. GO and KEGG analyses showed that wound repair and retinol metabolism-related pathways play an essential role in the molecular mechanism of COPD emphysematous phenotype. Three proteins, namely, KRT17, DHRS9, and FMO3, were selected for validation. While KRT17 and DHRS9 were highly expressed in the lung tissue samples of the COPD-E group, FMO3 expression was not significantly different between both groups. In conclusion, KRT17 and DHRS9 are highly expressed in the lung tissue of patients with COPD emphysematous phenotype. Therefore, these proteins might involve in wound healing and retinol metabolism in patients with emphysematous phenotype and can be used as phenotype-specific markers.

Keywords: DHRS9; KRT17; chronic obstructive pulmonary disease; emphysematous phenotype; proteomics.

FIGURE 1

Schematic diagram of tandem mass tag–based quantitative proteomic analysis. For the discovery cohort, every three tissue samples of COPD-NE patients were pooled together to form the three biological replicates in the COPD-NE group, and every four tissue samples of COPD-E patients were pooled together to form the three biological replicates in the COPD-E group. Liquid chromatography-tandem mass spectrometry analysis was performed for the discovery cohort. For the validation cohort, every lung tissue sample was used as an independent one. Western blot was performed to validate the results of liquid chromatography-tandem mass spectrometry analysis.

FIGURE 2

Quantification and hierarchical clustering of differential abundant proteins (DAPs) in COPD emphysmetous phenotype. (A) Volcano plot of DAPs. (B) Hierarchical clustering heat map of DAPs.

FIGURE 3

Gene ontology (GO) enrichment analysis of differential abundant proteins. (A) Top 20 enriched terms with smallest p values in GO enrichment analysis. (B) All level 2 GO terms involved in GO enrichment analysis.

FIGURE 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential abundant proteins. (A) Enriched terms in KEGG enrichment analysis with p values less than 0.05. (B) Top 20 KEGG terms with largest enriched protein numbers in KEGG enrichment results.

FIGURE 5

Protein–protein interaction (PPI) network of differential abundant proteins.

FIGURE 6

Type I cytoskeletal 17 (KRT17) and dehydrogenase/reductase SDR family member 9 (DHRS9) expressions are increased in the lung tissue samples of patients with chronic obstructive pulmonary disease emphysematous phenotype (COPD-E). (A) Western blotting results of KRT17, DHRS9, and FMO3 expressions in the lung tissue samples of COPD non-emphysematous phenotype (COPD-NE) and COPD-E groups. (B-D) Quantification of KRT17 (COPD-NE, n = 13; COPD-E, n = 24), DHRS9 (COPD-NE, n = 13; COPD-E, n = 24), and FMO3 (COPD-NE, n = 13; COPD-E, n = 25) protein expressions. **p < 0.01.