Proteomic profiling for plasma biomarkers of tuberculosis progression

Abstract

Severe pulmonary tuberculosis (STB) is a life‑threatening condition with high economic and social burden. The present study aimed to screen for distinct proteins in different stages of TB and identify biomarkers for a better understanding of TB progression and pathogenesis. Blood samples were obtained from 81 patients with STB, 80 with mild TB (MTB) and 50 healthy controls. Differentially expressed proteins were identified using liquid chromatography‑tandem mass spectrometry‑based label‑free quantitative proteomic analysis. Functional and pathway enrichment analyses were performed for the identified proteins. The expression of potential biomarkers was further validated by western blot analysis and enzyme‑linked immunosorbent assays. The accuracy, sensitivity and specificity for selected protein biomarkers in diagnosing STB were also evaluated. A total of 1,011 proteins were identified in all three groups, and 153 differentially expressed proteins were identified in patients with STB. These proteins were involved in 'cellular process', 'response to stimulus', 'apoptotic process', 'immune system process' and 'select metabolic process'. Significant differences in protein expression were detected in α‑1‑acid glycoprotein 2 (ORM2), interleukin‑36α (IL‑36α), S100 calcium binding protein A9 (S100‑A9), superoxide dismutase (SOD)1 in the STB group, compared with the MTB and control groups. The combination of plasma ORM2, IL‑36α, S100A9 and SOD1 levels achieved 90.00% sensitivity and 92.16% specificity to discriminate between patients with STB and MTB, and 89.66% sensitivity and 98.9% specificity to discriminate between patients with STB and healthy controls. ORM2, S100A9, IL‑36α and SOD1 were associated with the development of TB, and have the potential to distinguish between different stages of TB. Differential protein expression during disease progression may improve the current understanding of STB pathogenesis.

Figure 1.

Gene ontology annotations in terms of BP, CC and MF terms for the 153 differentially expressed proteins in plasma samples from patients with severe tuberculosis. BP, biological process; CC, cellular component; MF, molecular function.

Figure 2.

The enriched (A) upregulated (B) and downregulated signaling pathways for the 153 differentially expressed proteins in plasma samples from patients with severe tuberculosis. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3.

Visualization of protein-protein interaction network for dysregulated severe tuberculosis proteins. Dysregulated proteins are represented as spheres of distinct colors. Blue lines represent interactions between proteins and the thickness of the lines depicts the level of confidence associated with each interaction.

Figure 4.

Western blot analysis of eight selected proteins in plasma samples of the STB, MTB and NC groups. (A) Protein expression was determined by western blot analysis. (B) Densitometric analysis of the eight plasma proteins in the STB group compared with the MTB and NC groups. *P<0.05 and **P<0.01. N/S, not significant; STB, severe pulmonary tuberculosis; MTB, mild pulmonary tuberculosis; NC, healthy control samples; BTC, betacellulin; SOD, superoxide dismutase; ORM, α-1-acid glycoprotein; LYN, LYN proto-oncogene, Src family tyrosine kinase; IL-36α, interleukin-36α.

Figure 5.

Validation of ORM2, S100A9, IL-36α and SOD1 expression in plasma. Levels of these candidate biomarkers were measured by enzyme-linked immunosorbent assay in the NC (n=41), MTB (n=71), STB (n=72) groups. n, number of subjects. Median values are depicted by the red horizontal lines. *P<0.05 and **P<0.01. ORM2, α-1-acid glycoprotein 2; S100A9, S100 calcium binding protein A9; IL-36α, interleukin-36α; SOD, superoxide dismutase; STB, severe pulmonary tuberculosis; MTB, mild pulmonary tuberculosis; NC, healthy control samples.

Figure 6.

Efficacy of SOD1, S100A9, IL-36α and ORM2 in discriminating STB from MTB and the NC group. ROC curves of SOD1, S100A9, IL-36α and ORM2 in discriminating (A) STB from MTB, and (B) STB from the NC group. S100A9, S100 calcium binding protein A9; IL-36α, interleukin-36α; SOD, superoxide dismutase; ORM2, α-1-acid glycoprotein; STB, severe pulmonary tuberculosis; MTB, mild pulmonary tuberculosis; NC, healthy control samples; ROC, receiver operating characteristic.