Krisiukėnienė, Algirda

The global proteome of individual bronchial biopsy material from asthma and COPD patients has not been fully ascertained. The aim was to determine if mechanisms of disease and responses to treatment can be detected in biopsies from patients with asthma and COPD, using a quantitative proteomics technology. Endobronchial biopsies, pre and post treatment, were taken from patients with asthma (n=12) and COPD (n=11), as well as non-smoking (n=3) and smoking (n=2) healthy controls. Patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months). Quantitative proteomics technology was used to identify and quantify biopsy proteins. Pathways analysis was conducted to identify global proteome differences. A total of 1937 proteins were identified from all subjects. Proteome differences in asthma and COPD and changes in response to treatment could be observed. Analysis of the global proteome of COPD revealed that the top protein network contained the function of connective tissue disorder and that metabolic pathways were most relevant. In comparison, the top network in asthma contained dermatological functions, with the pathways being more actin-based. Changes in the proteome could also be observed in response to treatment. The increased relevance of cellular biological processes and decrease of oxidative stress can be seen for both asthma and COPD. Of particular interest, hepatic fibrosis was more associated with the COPD proteome and is altered with budesonide treatment. These results show that proteome differences can be detected using quantitative proteomics technology on clinically relevant biopsies from individual patients.

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 [...]

The global proteome of individual bronchial biopsy material from asthma and COPD patients has not been fully ascertained. The aim was to determine if mechanisms of disease and responses to treatment can be detected in biopsies from patients with asthma and COPD, using a quantitative proteomics technology. Endobronchial biopsies, pre and post treatment, were taken from patients with asthma (n=12) and COPD (n=11), as well as non-smoking (n=3) and smoking (n=2) healthy controls. Patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months). Quantitative proteomics technology was used to identify and quantify biopsy proteins. Pathways analysis was conducted to identify global proteome differences. A total of 1937 proteins were identified from all subjects. Proteome differences in asthma and COPD and changes in response to treatment could be observed. Analysis of the global proteome of COPD revealed that the top protein network contained the function of connective tissue disorder and that metabolic pathways were most relevant. In comparison, the top network in asthma contained dermatological functions, with the pathways being more actin-based. Changes in the proteome could also be observed in response to treatment. The increased relevance of cellular biological processes and decrease of oxidative stress can be seen for both asthma and COPD. Of particular interest, hepatic fibrosis was more associated with the COPD proteome and is altered with budesonide treatment. These results show that proteome differences can be detected using quantitative proteomics technology on clinically relevant biopsies from individual patients.

Background: Asthma and COPD are chronic lung diseases of high complexity. While studies have determined the involvement of many individual molecules and pathways, the global proteome of individual bronchial biopsies from asthma and COPD patients has not been fully ascertained. The aim of this study was to determine if mechanisms of disease and responses to treatment can be detected in biopsies from patients using quantitative proteomics and network analysis. Methods: Endobronchial biopsies, pre and post treatment, were taken from patients with asthma (n=12) and COPD (n=11), as well as non-smoking (n=3) and smoking (n=2) healthy controls. Patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months). Proteins (65 µg) extracted from the biopsies were digested and analysed using iTRAQ (isobaric tag for relative and absolute quantitation) combined with a nanoLC-LTQ-Orbitrap. Pathways analysis was performed on identified and quantified proteins using Ingenuity Pathways Analysis (IPA) to identify significant global proteome differences. Results: A total of 1937 proteins were identified from all subjects. Using IPA, proteome differences in asthma and COPD, as well as changes in response to treatment could be observed. Analysis of the global proteome of COPD revealed that the top network contained the function of connective tissue disorder and that metabolic pathways were most relevant. In comparison, the top network in asthma contained dermatological functions, while the pathways were more actin-based. An increased relevance of cellular biological processes and decrease in oxidative stress can be seen for both asthma and COPD in response to treatment. Of particular interest, fibrosis was more associated with the COPD proteome and is altered with budesonide treatment. Conclusion: These results show that proteome differences can be detected using quantitative proteomics.

Background: Asthma is a complex disease involving many intertwined processes. Many studies have determined the involvement of many individual molecules and pathways, but few have examined the global proteome of bronchial tissue in asthma, which can provide an improved understanding of ongoing biological processes. iTRAQ (isobaric tag for relative and absolute quantitation) with an LC-LTQ- Orbitrap is a sensitive, quantitative proteomics method. The aim was to determine if network analysis of quantitative proteomics of bronchial biopsies from asthmatics can reveal significant biological pathways, and whether these pathways are changed by inhaled glucocorticoid treatment. Method: Endobronchial biopsies were taken from untreated asthmatic patients (n=13) or non-smoking healthy controls (n=3). Asthmatics were randomised to double blind treatment with either placebo or budesonide (800 µg daily for 3 months) and new biopsies were obtained. Proteins (65 µg) extracted from the biopsies were digested and analysed using iTRAQ technology. Spectra obtained from nanoLC-MS/MS were used to identify and quantify proteins. Pathways analysis was performed on these proteins using Ingenuity Pathways Analysis (IPA). Result: The quantitative proteomics of bronchial biopsies successfully quantified 1814 proteins. When comparing asthma to healthy controls, the top canonical pathways identified by IPA for quantified proteins (min. fold change 1.5) included "acute phase response signalling", the "coagulation system", as well as the "intrinsic and extrinsic prothrombin activation pathways", with specific increases in the expression of fibrin and fibrinogen complexes, as well as kininogen. The effect of budesonide treatment on these interconnected pathways was examined using paired biopsy samples. While placebo treatment resulted in a decrease in the complexes, kininogen and some collagens, the budesonide treatment decreased the expression.

Background: Asthma is a disease involving many intertwined processes. Mouse models can be used to reflect features of asthma, but can never accurately reflect complex human processes, like remodelling. iTRAQ (isobaric tag for relative and absolute quantitation) with an LC-LTQ-Orbitrap is a sensitive, quantitative proteomics method. The aim was to determine if network analysis of quantitative proteomics of bronchial biopsies from asthmatics can reveal biological pathways and whether the pathways expressed are changed by inhaled glucocorticoid treatment. Method: Endobronchial biopsies were taken from untreated asthmatic patients (n=13) or non-smoking healthy controls (n=3). Asthmatics were randomised to double blind treatment with either placebo or budesonide (800 µg daily for 3 months) and new biopsies were obtained. Proteins (65 µg) extracted from the biopsies were digested and analysed using iTRAQ technology. Spectra obtained from nanoLC-MS/MS were used to identify and quantify proteins. Pathways analysis was performed on these proteins using Ingenuity Pathways Analysis (IPA). Result: The quantitative proteomics of bronchial biopsies successfully quantified 1814 proteins. Analysis of quantified molecules by IPA revealed that the top physiological system development and function categories included "tissue development" and "immune cell trafficking" for all compared groups. These molecules include two groups of proteins involved in epithelial repair, the laminins and collagens. While many of these proteins are undetectable in naive asthmatics compared to healthy subjects, treatment with budesonide increases the expression of laminin proteins compared to placebo treatment, which shows decreased expression. The collagen proteins show decreased expression with budesonide treatment compared to placebo. Conclusion: Analysis of the quantitative proteomics of bronchial tissue can quantify the proteins involved in the... [...].

Introduction. Recent proteomic studies on respiratory conditions investigate proteome changes to identify protein biomarker candidates for diagnosis, monitoring and treatment. A major challenge in clinical proteomics is the limited patient material. This is usually addressed by pooling patients into disease groups. With sensitive quantitative proteomics methods, like iTRAQ (isobaric tag for relative and absolute quantitation) technology, to identify and quantify proteomic profiles with mass spectrometry, it is possible to determine individual patient proteome differences without the need for pooling of biological material. The feasibility of this strategy is examined with the identification of proteins in bronchial biopsies of asthma and COPD patients, in an effort to identify disease and treatment specific protein biomarker candidates. Experimental. Endobroncial biopsies were taken from asthmatic (n=13) and COPD (n=17) patients, treted with budesonide or placebo, before and after treatment. Healthy subjects, smokers (n=3) and non-smokers (n=4) were used as controls. Biopsies were randomised over 20 sets of 4-plex experiments and the experiments run in a blinded manner. Each 4-plex experiment contained a common reference pool for normalisation purposes. The trypic peptides of the extracted protein were labelled with 4-plex iTRAQreagents and fractionated using cation exchange chromatography, before each of the fractions was subjected to reversed phase nanoLC-MS/MS. The resulting spectra were analysed by Proteome Discoverer and the Swissprot database was searched for protein identifications. Ingenuity Pathways Analysis was used to analyse the pathways and networks of differentially expressed proteins. [...].

Background: Recent proteomic studies on respiratory conditions investigate proteome changes to identify protein biomarker candidates for diagnosis, monitoring and treatment. A major challenge in clinical proteomics is the limited patient material. This is usually addressed by pooling patients into disease groups. With sensitive quantitative proteomics methods, like iTRAQ (isobaric tag for relative and absolute quantitation) technology, to identify and quantify proteomic profiles with mass spectrometry, it may be possible to determine individual patient proteome differences without the need for pooling. The feasibility of this strategy is examined with the identification of proteins in bronchial biopsies of asthma and COPD patients, in an effort to identify disease and treatment specific protein biomarker candidates. Methods: Bronchial biopsies per patient, per timepoint, were taken from asthmatic (n = 14) and COPD (n = 16) subjects, treated with budesonide or placebo, before and after treatment. Healthy subjects (n = 4) and healthy smokers (n = 4) were used as controls. Biopsies were randomised over 20 sets of 4-plex experiments with the operator being blinded to the disease status. Each 4-plex experiment contained a common reference pool for normalisation purposes. Proteins extracted from the biopsies were digested, labelled with iTRAQ reagents and fractionated using cation exchange chromatography, before each of the fractions was subjected to reversed phase nanoLC-MS/MS. The resulting spectra were analysed by Proteome Discoverer and the Swissprot database was searched for protein identifications. Results: Bronchial biopsies yielded a small, but sufficient amount of protein to conduct proteomic experiments using iTRAQ technology. [...].