Current Proteomics - Volume 8, Issue 2, 2011

Inflammation is a common pathological process underlying the most prevalent airway diseases worldwide. This process is characterized by abnormal leukocyte infiltration producing release of factors that induce vascular leakage, edema, smooth muscle cell contraction, mucous production, and generation of reactive oxygen species. Depending on the chemotactic factors produced by resident cells and the subtypes of T helper lymphocyte populations present, the infiltrating leukocytic cell types are quite distinct. Presently we understand that the initiation, maintenance and termination of airway inflammation involves intracellular innate signaling, expression of secreted bioactive proteins and effects of oxidation that affect resident cells within the pulmonary tissue. Here, proteomics technologies offer the potential for major new insights into these clinical syndromes. We have selected airway inflammation as the focus for this hot topic review because of the potential impact that proteomics approaches can have on the multiple facets of this complex clinical problem. The three major types of inflammatory airway disease discussed here are epidemic childhood wheezing (bronchiolitis), reactive airways disease (atopic asthma), and chronic obstructive pulmonary disease (COPD). Bronchiolitis is a lower respiratory tract infection of children, caused by the ubiquitous paramyxovirus, Respiratory Syncytial Virus. This virus causes acute mononuclear inflammation and may be an etiological factor in recurrent wheezing in childhood. Atopic asthma is a highly prevalent disease affecting up to 10% of the population in Western countries and accounts for >$3B annually in health care costs; this disease is characterized by eosinophilic inflammation and episodes of reversible obstruction provoked by non-specific allergen or viral infections. A specific form of asthma characterized by distinct neutrophilic inflammatory state is associated with a relative resistance to anti-inflammatory treatment. This syndrome is termed “severe” or “glucocorticoid resistant” asthma, and accounts for a disproportionately large share of asthma-related hospitalizations and significant reductions in quality of life. Finally, chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic airways inflammation and/or alveolar destruction. In this hot topic issue, we will illustrate current approaches (and future directions) of relevant discovery proteomics, detection of post-translational modifications, methods for analysis of large scale proteomic datasets, and approaches for focused biomarker assay development that are relevant to airway disease. The first review (Brasier) will review salient clinical features, mechanisms of innate signaling and highlight distinct types of inflammatory processes seen in these three types of airway disease. Critical to the proteomics-based exploration of these disease states is an understanding of the utility and limitations in the currently available sampling strategies for airway cells and proximal fluids. The second review (Wiktorowicz) will review important recent developments in discovery approaches and significant advances in protein quantification, including novel methods for detection of protein cysteinyl oxidation, increasingly recognized as the result of inflammatory, oxidative damage. The third review (Pazdrak) will discuss the insights gleaned into proteomics in the major leukocyte populations known to be important in the initiation and maintenance of asthma. The fourth review (Sadygov) will update the rapidly changing bioinformatics tools available for global (“bottoms-up”) mass spectrometry experiments including stable isotopic labeling and label-free proteomics. Finally, the fifth review (Zhao) will discuss methods for biomarker assay development using selective reaction monitoring. Properly applied, these proteomics approaches will extend our understanding of the mechanisms, as well as impact therapy by identification of physiological distinct subsets of airway inflammatory disease.

Airways disease presenting as viral bronchiolitis, asthma and chronic obstructive pulmonary disease (COPD) is an increasingly important source of morbidity in Western Countries. Although these diseases affect distinct age groups, have different initiators and manifest variable amounts of parenchymal remodeling, each process is driven by a common fundamental process involving cellular inflammation. Inflammation is a coordinated multi-cellular response to the presence of pathogens or allergens culminating in altering resident leukocyte populations and remodeling structural tissues in the lung. In this review, we will discuss the contributions of innate and chronic adaptive immunity underlying inflammation seen in bronchiolitis, asthma, both mild and severe, and COPD. We will illustrate how applications of proteomics have begun to provide new insights into critical questions regarding the cellular and innate response to inflammation, role of reactive oxygen stress, identification of phenotypic subgroups using molecular profiling, and how proteomics provides insights into therapeutic responses. We will discuss the available sampling strategies for airway biofluids ( bronchoalveolar lavage, induced sputum, exhaled bronchial breath condensates, and nasopharyngeal aspiration) and the advantages and limitations of each. Selectively applied and properly designed proteomics studies provide an added dimension of information to reduce the impact and burden of these common inflammatory airway diseases.

Discovery proteomics is defined here as the unbiased (untargeted) differential investigation of protein abundances from biological material resulting from some biological or physical challenge. In this review, we will examine the latest approaches and strategies for discovery investigations of airway disease. We will discuss general and specific sampling strategies, sample preparation, differential separations and quantification—with special attention to posttranslational modifications that are particularly relevant to oxidative stress response of airway tissues, statistical analysis and tools, mass spectrometric identification, and finally bioinformatics tools for pathway analysis.

Considerable research indicates that many cells of the asthmatic airway become dysfunctional. There is clear evidence for epithelial and airway smooth muscle cell dysfunction, accumulation of activated T cells, and systemic inflammation effects on peripheral blood granulocytes. A number of studies of airway cells have applied proteomics, resulting in mapped protein profiles of inflammatory cells and increased understanding of potential mechanisms involved in allergic inflammation. The reports described in this review offer many encouraging leads for further detailed analysis of allergic processes. The elucidation of their role in asthma will require the application of novel technologies and insightful biochemical interpretation. There is considerable optimism that comparative proteomic investigation of airway inflammatory cells may provide critical insights into the phenotypes of asthma and will reveal novel mechanistic targets for therapeutic interventions.

Determining global proteome changes is important for advancing a systems biology view of cellular processes and for discovering biomarkers. Liquid chromatography, coupled to mass spectrometry, has been widely used as a proteomics technique for discovering differentially expressed proteins in biological samples. However, although a large number of high-throughput studies have identified differentially regulated proteins, only a small fraction of these results have been reproduced and independently verified. The use of different approaches to data processing and analyses is among the factors which contribute to inconsistent conclusions. This paper provides a comprehensive and critical overview of bioinformatics methods for commonly used mass spectrometry-based quantitative proteomics, employing both stable isotope labeling and label-free approaches. We evaluate the challenges associated with current quantitative proteomics techniques, placing particular emphasis on data analyses. The complexity of processing and interpreting proteomics datasets has become a central issue as sensitivity, mass resolution, mass accuracy and throughput of mass spectrometers have improved. We review a number of computer programs designed to address these challenges. We focus on approaches for signal processing, noise reduction, and methods for protein abundance estimation.

Recent advances in global-scale proteomic technology enable identification of hundreds of candidate biomarkers. However, very few candidates so identified can reach the high bar of FDA approval for clinical use. The low efficiency of biomarker approval reflects the challenges of taking candidate biomarkers identified in discovery research through the long and difficult pipeline required for biomarker development. The greatest challenge in biomarker development is the lack of reliable assays for use in the verification and validation phases. This paper reviews methodologies and challenges for biomarker assay development with emphasis on stable isotope dilution coupled with multiple reaction monitoring-mass spectrometry (SID-MRM-MS). Because of its sensitivity, quantification abilities, and specificity, SIDMRM- MS has the potential to bridge the critical rate-limiting gaps between the biomarker discovery- and validation phases. A workflow for generation of a specific SID-MRM-MS assay is presented. We conclude that currently, SIDMRM- MS assay is a promising technology for biomarker verification and validation. To move the technology toward an FDA-approvable platform, more stringent evaluation must be performed and these future studies will require a joint effort of the clinical proteomics community, the regulatory agency and major mass spectrometer manufacturers.

Recent developments in mass spectrometry have brought clinical proteomics to the forefront of cancer diagnosis and treatment, offering reliable, robust and efficient analysis methods for biomarker discovery and monitoring. Proteins control cellular processes through intricate signaling cascades and protein function is often determined by posttranslational modifications. However, alteration of post-translational modifications that control normal cellular processes, such as differentiation, proliferation and apoptosis, has been significantly related to tumorigenesis. Aberrations in protein phosphorylation, for example, offer promising opportunity for disease diagnosis and prognosis and could also serve as a therapeutic indicator for cancer treatment. Recent studies have identified biomarkers involving several cancer types using high-performance liquid chromatography separation and enrichment techniques coupled with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry for protein identification and quantification. However the clinical implementation of proteomic technologies is not without shortcomings and is still dependent on laboratory research to expand identification of relevant biomarkers.