Beyond fishing: the role of discovery proteomics in mechanistic lung research

the field of discovery proteomics has grown exponentially over the past 10 years. A variety of methods have been developed and refined for identifying proteins in complex biological samples (7), and access to these methodologies has become widespread. Sensitivity and reproducibility are improving steadily as are the statistical tools, analytical software, and protein databases necessary for analysis and interpretation of large proteomic datasets (5). Given the increasing number of studies that utilize proteomic discovery methodologies (9), it is important to reflect on the potential role of discovery proteomics in mechanistic studies in lung disease to address the following questions. As discovery proteomic methodologies move from novel methods to stable laboratory techniques, what should the standards for publication of discovery proteomic studies be in The American Journal of Physiology Lung Cellular and Molecular Physiology? Are we poised to move beyond descriptive lists of differentially expressed proteins into a new era of mechanistic proteomic studies?

Proteomic studies of human samples have largely yielded descriptive lists of differentially expressed proteins. Although these studies have potential value for identification of biomarkers of lung disease for clinical use (5), to date they have not yielded substantial mechanistic insight. In an example of an early application of proteomic methodologies to lung disease, Bowler and colleagues (2) used protein profiling by two-dimensional electrophoresis followed by mass spectrometry to identify proteins that were differentially expressed in bronchoalveolar lavage (BAL) from patients with acute lung injury compared with normal controls. In addition to changes in levels of many BAL proteins in acute lung injury compared with controls, they also identified a number of posttranslational protein modifications characteristic of acute lung injury. In a more recent study of serial BAL from patients with acute lung injury, Chang and colleagues (4) applied principal component analysis, gene ontology analysis, and network analysis to proteins identified by two-dimensional gel electrophoresis, illustrating the major advances in analytical tools for proteomic studies that have occurred in the past decade. In addition to differential expression of proteins in the BAL over time in ARDS compared with controls, they identified several specific biological pathways that were most highly affected in acute lung injury including immunity, defense responses, and inflammatory responses to pathogens and injury. Network analysis further identified several nodes within the complex protein interactome that might represent therapeutic targets, thereby moving closer to the mechanistic insight that would be the ideal result of application of proteomic methodologies to human lung diseases.

Proteomic studies of cell culture systems allow controlled studies of experimental manipulations that affect the cellular or secreted proteome. In a recent study published in AJP-Lung, Candiano and colleagues (3) analyzed the proteome of airway surface liquid secreted by primary cultures of normal human bronchial epithelial cells grown on an air-liquid interface and identified a number of proteins that were differentially secreted after stimulation by proinflammatory cytokines, including several proteins with putative antibacterial activity and a number of proteases and antiproteases. They also identified different patterns of change in protein expression resulting from stimulation with interleukin-4 compared with interleukin-1β, tumor necrosis factor-α, or interferon-γ (3). This type of in vitro study has the advantage of highly controlled conditions that facilitate identification of differentially expressed proteins, but extrapolation to complex in vivo systems is limited.

Proteomic discovery methodologies have also been applied to in vivo experimental systems. Hirsch and colleagues (8) assessed the cellular proteome of rat alveolar epithelial type II cells that were isolated from rats that underwent ischemia and reperfusion of the liver compared with sham-treated rats using liquid-chromatography followed by electrospray tandem mass spectrometry. Differential expression of a number of proteins in type II cells in response to hepatic ischemia reperfusion was identified including catalase and enzymes in ATP-generating pathways. Recently, Umstead and colleagues (11) used two-dimensional gel electrophoresis followed by mass spectrometry to study changes in BAL proteins between juvenile and aged rats. In addition to changes in protein levels with age, increases in oxidation status of proteins were found with aging, particularly in proteins with functions in host defense, redox regulation, and protein metabolism. The authors (11) speculate that age-related increases in protein oxidation may compromise host defense function in aged rats, but this hypothesis was not further tested.

In another study published in AJP-Lung in 2008 (1), data from a proteomic study was used to identify a potential mechanistic pathway in pulmonary fibrosis. Bogatkevich and colleagues (1) compared the cellular proteome of normal human lung fibroblasts transfected with the profibrotic cytokine connective tissue growth factor (CTGF) to control fibroblasts and to lung fibroblasts isolated from patients dying with advanced scleroderma-related pulmonary fibrosis. Using two-dimensional gel electrophoresis followed by in-gel digestion and mass spectroscopy, they identified 13 proteins that were upregulated by transfection of fibroblasts with CTGF, including 9 novel proteins that have not been previously associated with pulmonary fibrosis; 6 of these proteins were also upregulated in lung fibroblasts from scleroderma patients. In a series of targeted experiments, they then established a potential mechanism whereby one of the proteins induced by CTGF, IQ motif containing GTPase activating protein (IQGAP1), may contribute to pulmonary fibrosis. Using siRNA to knock down IQGAP1 in both normal and scleroderma lung fibroblasts, they found that IQGAP1 plays an important role in fibroblast migration in an in vitro wound-healing assay, an effect that was mediated in part through MAP kinase signaling pathways. This study established a new paradigm for integration of discovery proteomic methodologies into mechanistic lung research, moving beyond mere identification of differentially expressed proteins to mechanistic experimental studies.

Looking to the near future, what should be our goal for publication of discovery proteomic studies in AJP-Lung? First, studies should adhere to published guidelines for reporting of discovery proteomics (10) and gel electrophoresis experiments (6). Second, to be of maximum interest and utility to the readership of this journal, we must push for extension of findings from mere descriptive lists to actual mechanistic studies. Only when the findings of discovery proteomic studies are used to shed light on the mechanisms of acute and chronic lung disease will we begin to realize the potential of these new methodologies. Eventually, this approach may yield new therapeutic strategies for pulmonary diseases.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-081332 and HL-51856.