Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics.
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ABSTRACT:
SUBMITTER: Knott SJ
PROVIDER: S-EPMC7961849 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.
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Project description:BACKGROUND:The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. METHODS:We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS:Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION:The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING:UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.
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Project description:Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days postamputation) time point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.
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