| Summary: | 博士 === 國立臺灣師範大學 === 化學系 === 96 === Many membrane proteins are implicated in particular diseases states and often are attractive therapeutic targets. Comprehensive and quantitative profiles of membrane proteins facilitate our understanding of their roles in regulating biological processes and in cellular signaling. Towards multiplexed, comprehensive and robust quantitation of the membrane proteome, we developed a strategy combining gel-assisted digestion, iTRAQ labeling, and LC-MS/MS. The first part of thesis focus on the methodology development and performance evaluation of the new strategy. The gel-assisted digestion offers advantages of diverse compatibility with high concentrations of detergent and salts for efficient solubilization, denaturation and digestion of membrane proteins. This new digestion method was applied to the proteomic profile of bronchoalveolar lavarge fluid (BALF) from patients with ventilator-associated pneumonia (VAP). The data obtained from the cohort of VAP patients demonstrated that our approach provides in-depth description of the BALF proteome. The gel-assisted digestion also improved identification and quantitation accuracy by peptide-level isotopic tagging of amino groups. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (< 8% error) and precision (< 12% RSD), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane protein—each quantified based on average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that previously achieved using MS and provides superior quantitation accuracy compared with other methods.
In the second part of thesis, the new strategy was applied to two biology systems. The first example presents the first proteomic delineation of phenotypic expression in a mouse model of autosomal-dominant polycystic kidney disease (ADPKD) tissues. Although ADPKD is the most prevalent and potentially lethal inherited human renal disease, systematic mapping of phenotypic expression of the disease and uncovering perturbed cellular networks remain to be further investigated. By characterizing kidney cell plasma membrane from wild-type versus PKD1 knockout mice, 791 proteins were quantified and 67 and 37 proteins showed ≥ 2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.
The second example focuses on the human colorectal cancer (CRC). Colorectal cancer is a worldwide disease and it is equally common in both men and women. Quantitative analysis of differentially expressed membrane proteins in the paired normal and cancerous human colorectal cancer (CRC) tissues will provide opportunity for discovery of biomarker candidates. The preliminary data shows that 304 proteins were quantified from 8 patients in Dukes’ A, B, C, and D stages; 39 proteins show ≥ 2-fold differentially expression in all the eight patients. The identified up-regulation of carcinoembryonic antigen-related cell adhesion molecule 6 and 5 (CEA) in Dukes’ B and C patients were consisted with previous literatures on their roles as biomarker of colorectal cancer. Several secreted proteins and plasma membrane proteins, that might be potential diagnostic biomarkers or drug targets, were also quantified in this study.
Taken together, this multiplexed quantitation platform provides a generic and powerful complement to membrane proteomics. We expect that the approach can be a generic strategy to investigate differential expression of membrane proteins in many sample types including cells, tissues and body fluids under different environmental and pathophysiological conditions to discover membrane protein markers or to delineate the pathogenesis of certain diseases.
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