Hansen Kirk C, Kiemele Lauren, Maller Ori, O'Brien Jenean, Shankar Aarthi, Fornetti Jaime, Schedin Pepper
University of Colorado Cancer Center Proteomics and Mass Spectrometry Facility, Department of Pediatrics, University of Colorado Denver, Aurora, Colorado 80045, USA.
Mol Cell Proteomics. 2009 Jul;8(7):1648-57. doi: 10.1074/mcp.M900039-MCP200. Epub 2009 Apr 7.
Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.
上皮细胞行为由周围细胞外基质(ECM)的组成协调;因此,ECM蛋白鉴定对于理解正常生物学和疾病状态至关重要。ECM的不溶性和抗消化性阻碍了对ECM蛋白的蛋白质组学分析。在这里,我们探索将快速超声处理和表面活性剂辅助消化相结合用于ECM样品详细蛋白质组学分析的效用。与传统的过夜消化相比,这种优化方法显著提高了胶原蛋白I的序列覆盖率,揭示了基质胶中数百种先前未鉴定蛋白质的存在,并确定了从大鼠乳腺分离的ECM的蛋白质谱,该蛋白质谱与基质胶中的蛋白质谱有很大不同。在一项研究上皮细胞与ECM相互作用的三维培养试验中,发现与基质胶相比,乳腺上皮细胞接种乳腺来源的基质时会发生广泛的分支形态发生。这些数据累积起来突出了ECM组成和功能的组织特异性,并强调需要优化技术,如本文所述的技术,用于ECM样品的蛋白质组学表征。