© 2006 American Thoracic Society doi: 10.1164/rccm.2602001
The Promising Future of Proteomics in SarcoidosisPadua University School of Medicine, Padua, Italy Proteomic technologies are widely used to identify biomarkers for the early detection of human diseases, to develop predictive models of classification, and for monitoring the therapeutic efficacy of drugs. In this issue of the AJRCCM (pp. 1145–1154), Kriegova and colleagues (1) used an innovative proteomic technique, surface-enhanced laser desorption ionization–time-of-flight mass spectrometry (SELDI-TOF MS) to examine patterns of protein expression in sarcoidosis. Mass spectrometry–based techniques may be considered a direct derivation of the original studies on proteomics by Hutchens and Yip (2). Combining chromatography and mass spectrometry, this platform is capable of detecting tens of thousands of data points in a short time from very small volumes (microliters) of biofluids containing a complex mixture of peptides or proteins, such as urine, serum, plasma, or bronchoalveolar lavage (BAL). The methodology resolves proteins, present in mixtures even at extremely low concentrations (femto- to attomoles), through a high-throughput approach (300 samples/day in batches of 100) (3). The principle behind the technique is very simple (Figure 1), with proteins of interest, such as those contained in the BAL, captured on an immobilized stationary-phase chip in an array format. The chip is then inserted into a vacuum chamber and bound proteins are irradiated with a laser that results in the molecules liberating gaseous ions. The ions are rapidly accelerated into a "flight tube" and the time of flight, which is related to the mass-to-charge ratio (m/z) of each protein/peptide, allows for their separation. An ion trap analyzer functions as a detector, which, when capturing the ions, starts to oscillate and transforms ion cyclotron resonance into signals. Detected proteins and peptides are then shown as a series of corresponding peaks. Since a typical mass spectrometry platform may generate a proteomic profile of up to 400,000 data points, sophisticated software packages are needed for the statistical analysis of large numbers of samples.
The on-chip capture and detection of proteins have evident advantages; it allows small sample sizes and limited sample manipulation, and produces high-throughput results (3, 4). Taking advantage of these advances, mass spectrometry platforms have been used for the discovery of biomarkers in a large variety of diseases, including cancer and autoimmune disorders, as well as in neurologic and metabolic disorders (3). The tool is also beginning to be used in respiratory medicine. Zhukov and colleagues (5) utilized SELDI-TOF MS to generate specific protein peak patterns for malignant lung tumors, whereas Merkel and colleagues identified specific protein patterns in BAL fluid samples of patients with chronic obstructive pulmonary disease (6). The article published in this issue of the Journal represents an example of the potential utility of SELDI-TOF MS. The authors used this technique to examine the BAL proteome in patients with sarcoidosis. Data analysis identified a panel of 40 proteins that were altered in samples with this disorder versus those from healthy controls. Distinct protein patterns specific for patients with pulmonary sarcoidosis were identified and, more specifically, profiles were characterized from patients with sarcoidosis and Löfgren's syndrome. The authors also found distinguishing peaks on SELDI-TOF MS that correlated with the radiologic stages of the disease. After reverse-phase fractionation, peptide fingerprint mapping, and immunodepletion, a novel protocadherin-2 precursor was identified in the context of the sarcoidosis proteome. The potential clinical utilities of such an approach are clear. Refinement of the characterization of the sarcoid proteome is likely to help to identify molecules expressed in different phases of the disease, which might, at least in theory, represent putative molecular targets. Furthermore, a proteomic profile could be designed for the clinical evaluation of patients with sarcoidosis. As protein expression data become available, the integration of this technology into protocols for disease diagnosis as well as outcome prediction should soon take place in sarcoidosis. Finally, the application of proteomics could be used in verifying the in vitro effect of molecules, a crucial step in the development of therapeutics. For instance, levels of immunosuppressive or immunomodulatory molecules could be measured directly from a tissue biopsy to assess adequacy of drug delivery to a particular organ site involved by the sarcoid process. Beyond its clinical implications, proteomic approaches might yield novel information about the pathophysiology of the disease. For example, using SELDI-TOF MS, a number of differences in protein patterns have been demonstrated between alveolar macrophages (AMs) and their precursors or among AMs under different pathologic conditions (7). Characterization of macrophage-derived and Th1-derived proteins during sarcoidosis will offer a better understanding of its pathogenesis. Laser-capture microdissection permits the isolation of single cells or single populations of cells from thin tissue sections. In combination with SELDI-TOF MS, laser-capture microdissection promises to permit the collection of protein signatures from a single cell type within a heterogeneous sample obtained from a tissue involved by sarcoidosis (8). This information could significantly improve the understanding of molecular mechanisms underpinning granuloma formation and impact diagnostic pathology of the disease. It is easy to anticipate that within 5 years the clinical application of proteomic-based techniques will have transformed our knowledge of most physiologic and pathologic pulmonary processes, including sarcoidosis. To use such methodology on a routine basis for a protein-based evaluation of patients with sarcoidosis, the clinical sensitivity and specificity of protein profiling of BAL should be validated by different groups. Of note, comparative analyses of proteomic data obtained from biofluids highlighted important discrepancies in the final results among laboratories, mostly due to the absence of standardized sample collection and processing, and in the use of different pre- and postanalytic strategies (9, 10). For these reasons, the international scientific community is adopting standardized protocols, which include the use of pooled reference standard samples applied on each protein array and standardized daily calibrations of the instruments, as the minimal essential conditions to validate the data produced by SELDI. There is also a clear need to develop international standards for archiving data obtained from the analysis of lung samples. The validation of standardized SELDI-TOF MS methodologies will perhaps require months or years of cooperative effort by the groups involved, but the result will be a rich source of information to aid the physician in patient management. Acknowledgments The authors thank Dr. Martin Donach for his help in the preparation of the manuscript. FOOTNOTES Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. REFERENCES
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