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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Endobronchial biopsies were taken from untreated asthmatic patients (<it>n </it>= 12) and healthy controls (<it>n </it>= 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p> <p>Trial registration</p> <p>ClinicalTrials.gov registration <a href="http://www.clinicaltrials.gov/ct2/show/NCT01378039">NCT01378039</a></p

Mass spectrometry-based proteomic strategies applied to Helicobacter pylori. A Search for candidate vaccine antigens

This thesis describes the development and refinement of proteomic strategies and the application of these on the human gastric pathogen H. pylori. The purpose was to identify proteins, and in particular membrane proteins, as potential targets for vaccine development. A semi-preparative method based on liquid-phase separation of whole cell extracts was found to be useful. However, in order to perform a more comprehensive analysis of the outer membrane protein profile, and to identify proteins specific for disease-related H. pylori strains, a new strategy combining subcellular fractionation with high-sensitivity nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) analysis was used. Differences among outer membrane proteins (OMPs) were compared in clinical bacterial isolates of asymptomatic carriers and of patients with duodenal ulcer or gastric cancer. Several potential targets for a vaccine were identified and proteins that may be used as diagnostic markers or should be subjected to more detailed studies were identified. These results document the importance of using nano-LC coupled to the powerful FT-ICR MS method for high confidence identification of low-abundance components of a complex mixture. The protein expression patterns of the mouse-adapted H. pylori strain SS1 and its isogenic HpaA mutant strain were examined using the two strategies. Whole cell extracts were analyzed by traditional 2D- gel electrophoresis (2D-GE) and differential in-gel electrophoresis (DIGE) technology, and the OMP profiles were compared using the fractionation and nano-LC MS approach. Only minor differences in the protein expression pattern were found between the two strains. HpaA was identified in the SS1 strain, but could not be detected in the mutant strain, although the low-abundance OMP BabA was readily identified. Infection studies in mice demonstrated that HpaA is essential for H. pylori colonization, and this protein is therefore a candidate vaccine antigen. In order to evaluate the potential of using mass spectrometry to localize surface-exposed peptides of a protein (potential immunogenic epitopes), we performed chemical cross-linking studies of the urease complex of H. pylori. This protein is easy to prepare and the known three-dimensional structure facilitates the interpretation of results by molecular modeling. Although cross-linked peptides were identified, the conclusion is that this potentially helpful technique requires additional developmental efforts to be practically useful

HpaA Is Essential for Helicobacter pylori Colonization in Mice

Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice

Mergers and acquisitions as a way to bank restructuring

Теоретичні основи та проблеми процесів злиття та поглинання банків розглянуто в наукових працях багатьох сучасних дослідників. Так, окремі теоретичні та практичні аспекти досліджували вітчизняні науковці О. О. Чуб, З. М. Васильченко, М. І. Диба, В. І. Міщенко, А. В. Шаповалов. Проте, незважаючи на велику кількість наукових розробок з даної проблематики, внаслідок світової фінансової кризи 2007–2009 років, процеси злиття та поглинання потребують подальшого дослідження. Насамперед це стосується питань, пов’язаних із дослідженням сутності й особливостей проведення структурної реорганізації банків у вітчизняних умовах

Strain-Level Typing and Identification of Bacteria Using Mass Spectrometry-Based Proteomics

Because of the alarming expansion in the diversity and occurrence of bacteria displaying virulence and resistance to antimicrobial agents, it is increasingly important to be able to detect these microorganisms and to differentiate and identify closely related species, as well as different strains of a given species. In this study, a mass spectrometry proteomics approach is applied, exploiting lipid-based protein immobilization (LPI), wherein intact bacterial cells are bound, via membrane-gold interactions, within a FlowCell. The bound cells are subjected to enzymatic digestion for the generation of peptides, which are subsequently identified, using LC–MS. Following database matching, strain-specific peptides are used for subspecies-level discrimination. The method is shown to enable a reliable typing and identification of closely related strains of the same bacterial species, herein illustrated for <i>Helicobacter pylori</i>