Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment

Abstract

Background: Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions.

Methods: Endobronchial biopsies were taken from untreated asthmatic patients (n = 12) and healthy controls (n = 3). Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months) and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment.

Results: More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo.

Conclusions: Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be identified using this approach and the expression of these features is changed by inhaled glucocorticoid treatment. Quantitative proteomics may be applied to identify mechanisms of disease that may assist in the accurate and timely diagnosis of asthma.

Trial registration: ClinicalTrials.gov registration NCT01378039.

Figures

Figure 1

Schematic overview of the experimental workflow. The workflow used in the proteomics analysis of human endobronchial biopsies. Proteins were extracted and digested, before the peptides were labelled with iTRAQ® Reagents in a four-plex set. Each four-plex, containing one reference pool and three samples, were fractionated using ion exchange chromatography. Each fraction was then subjected to nano LC-MS/MS. The resulting spectra were searched for identification and quantification. The identified and quantified proteins were then analysed using Ingenuity Pathways Analysis.

Figure 2

FENO and FEV1% predicted of healthy controls and asthmatics. The scatter plots show the airway inflammation and lung function of healthy controls (n = 3) and untreated asthmatics (n = 12) as assessed by FEV1% predicted (A) and fractional exhaled nitric oxide (FENO) (B). Asthmatics had significantly increased airway inflammation and significantly decreased lung function, compared to healthy controls. The bars indicate the mean ± SEM. * = indicates p value < 0.05.

Figure 3

Network of differentially expressed proteins in untreated asthmatics compared to healthy controls. The top network generated by Ingenuity Pathways Analysis from quantified proteins (1.5 fold change threshold) of untreated asthmatics compared to healthy controls, contains the functions of haematological system development and function, lipid metabolism and molecular transport. Molecules with bold outline are significantly different between the two groups using a Student's t-test. Lines indicate relationships between molecules. Arrows at the end of these lines indicated the direction of the interaction. Molecules that are up-regulated and down-regulated in the dataset are coloured red and green respectively. Grey molecules do not meet the cut-off threshold. Uncoloured molecules have been added from the Ingenuity Knowledge Base. Entrez gene names for molecules are: ACAT1 = acetyl-CoA acetyltransferase 1, APOA1 = apolipoprotein A-I, APOA2 = apolipoprotein A-II, CA2 = carbonic anhydrase II, CAP1 = CAP, adenylate cyclase-associated protein 1 (yeast), CS = citrate synthase, GLO1 = glyoxalase I, GOLGA3 = golgin A3, HBB = hemoglobin (beta), HBD = hemoglobin (delta), HBG1 = hemoglobin (gamma A), HLA-B = major histocompatibility complex, class I, B, HNRNPH1 = heterogeneous nuclear ribonucleoprotein H1 (H), MVP = major vault protein, MYLK = myosin light chain kinase, NONO = non-POU domain containing, octamer-binding, PAK2 = p21 protein (Cdc42/Rac)-activated kinase 2, RAB14 = RAB14, member RAS oncogene family, RPL8 = ribosomal protein L8, SYNCRIP = synaptotagmin binding, cytoplasmic RNA interacting protein, TAGLN2 = transgelin 2, TARDBP = TAR DNA binding protein, TNS1 = tensin 1, TPM3 = tropomyosin 3, UQCRB = ubiquinol-cytochrome c reductase binding protein, UQCRC2 = ubiquinol-cytochrome c reductase core protein II, VASP = vasodilator-stimulated phosphoprotein.

Figure 4

Network of differentially expressed proteins of post placebo compared to paired pre placebo biopsy. The top network generated by Ingenuity Pathways Analysis from quantified proteins of post placebo asthmatics compared to their pre placebo biopsy. The network contains the functions of hair and skin development and function, organ development, dermatological diseases and conditions. Molecules with bold outline are significantly different between the two groups using a Student's t-test. Lines indicate relationships between molecules. Arrows at the end of these lines indicated the direction of the interaction. Molecules that are up-regulated and down-regulated in the dataset are coloured red and green respectively. Grey molecules do not meet the cut-off threshold. Uncoloured molecules have been added from the IPA KB. Entrez gene names for molecules are: CFL1 = cofilin 1 (non-muscle) DES = desmin, E2F7 = E2F transcription factor 7, HDGF = hepatoma-derived growth factor, HRG = histidine-rich glycoprotein, IGHG3 = immunoglobulin heavy constant gamma 3 (G3 m marker), IGHG4 = immunoglobulin heavy constant gamma 4 (G4 m marker), IGKC = immunoglobulin kappa constant, KRT2 = keratin 2, KRT4 = keratin 4, KRT5 = keratin 5, KRT8 = keratin 8, KRT9 = keratin 9, KRT13 = keratin 13, KRT14 = keratin 14, KRT15 = keratin 15, KRT17 = keratin 17, KRT19 = keratin 19, KRT6A = keratin 6A, PPIF = peptidylprolyl isomerase F, PPL = periplakin, S100P = S100 calcium binding protein P, SERPINA4 = serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 4, TACSTD2 = tumor-associated calcium signal transducer 2, TMOD2 = tropomodulin 2 (neuronal), TNFSF13B = tumor necrosis factor (ligand) superfamily, member 13b, TPI1 = triosephosphate isomerase 1, TPM1 = tropomyosin 1 (alpha).

Figure 5

Network of differentially expressed proteins of post budesonide compared to paired pre treatment biopsy. The top network generated by Ingenuity Pathways Analysis from quantified proteins of post budesonide asthmatics compared to their pre placebo biopsy. The network contains the functions of cancer, genetic disorder and respiratory disease. Molecules with bold outline are significantly different between the two groups using a Student's t-test. Lines indicate relationships between molecules. Arrows at the end of these lines indicated the direction of the interaction. Molecules that are up-regulated and down-regulated in the dataset are coloured red and green respectively. Grey molecules do not meet the cut-off threshold. Uncoloured molecules have been added from the IPA KB. Entrez gene names for molecules are: CAPNS1 = calpain, small subunit 1, CAST = calpastatin, CFL1 = cofilin 1 (non-muscle), CKB = creatine kinase, brain, DPYSL2 = dihydropyrimidinase-like 2, DPYSL3 = dihydropyrimidinase-like 3, DPYSL5 = dihydropyrimidinase-like 5, DYNLL2 = dynein, light chain, LC8-type 2, ENO1 = enolase 1, (alpha), GPI = glucose-6-phosphate isomerase, H2AFX = H2A histone family, member X, HNRNPD = heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding protein 1, 37 kDa), HYOU1 = hypoxia up-regulated 1, ILF3 = interleukin enhancer binding factor 3, 90 kDa, KRT4 = keratin 4, KRT9 = keratin 9, KRT6A = keratin 6A, LDHA = lactate dehydrogenase A, PPP1CC = protein phosphatase 1, catalytic subunit, gamma isozyme, RPL31 = ribosomal protein L31, SERBP1 = SERPINE1 mRNA binding protein 1, SYNCRIP = synaptotagmin binding, cytoplasmic RNA interacting protein, TTR = transthyretin, VIM = vimentin, XRCC5 = X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining), YWHAE = tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide, YWHAZ = tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide.