An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage
An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage
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DOI：
10.1074/mcp.m900039-mcp200
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发表时间：
2009-07-01
影响因子：
7
通讯作者：
Schedin, Pepper
中科院分区：
文献类型：
--
作者：
Hansen, Kirk C.;Kiemele, Lauren;Schedin, Pepper
上皮细胞的行为是由周围的细胞外基质（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蛋白的蛋白质组学分析受到ECM的不溶性和耐消化性质的阻碍。在这里，我们探索了快速超声处理和表面活性剂辅助消化相结合用于ECM样本详细蛋白质组学分析的实用性。与传统的过夜消化相比，这种优化的方法显着提高了胶原蛋白I的序列覆盖率，揭示了数百个以前未识别的蛋白质在基质胶中的存在，并确定了从大鼠乳腺中分离的ECM的蛋白质谱，这与基质胶中发现的有很大不同。在研究上皮细胞-ECM相互作用的三维培养试验中，发现与Matrigel相比，用乳腺衍生基质铺板时，乳腺上皮细胞经历了广泛的分支形态发生。累积这些数据突出了ECM组成和功能的组织特异性性质，并强调需要优化技术，如本文所述的那些，用于ECM样品的蛋白质组学表征。Molecular & Cellular Proteomics 8：1648-1657，2009.
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. Molecular & Cellular Proteomics 8: 1648-1657, 2009.