Proteornics discovery of metalloproteinase substrates in the cellular context by iTRAQTM labeling reveals a diverse MMP-2 substrate degradome
Proteornics discovery of metalloproteinase substrates in the cellular context by iTRAQTM labeling reveals a diverse MMP-2 substrate degradome
复制标题
DOI：
10.1074/mcp.m600341-mcp200
复制
发表时间：
2007-04-01
影响因子：
7
通讯作者：
Overall, Christopher M.
中科院分区：
文献类型：
--
作者：
Dean, Richard A.;Overall, Christopher M.
阐明蛋白水解酶底物降解域对于了解蛋白水解酶网络中蛋白分解途径的功能以及蛋白水解酶如何调节细胞功能是至关重要的。我们利用一种新的多重蛋白质组学方法，鉴定了基质金属蛋白酶-2(MMP2)裂解蛋白，溶解了细胞周围基质，并在细胞环境中去除了细胞外结构域。从MMP2(-/-)成纤维细胞条件培养液中收集不同时间点低水平表达活性人基质金属蛋白酶-2的完整蛋白和裂解蛋白的胰酶多肽，用iTRAQ(TM)质量标签进行胺标记。在串联MS过程中，通过标记裂解实现了多肽鉴定和活性和非活性蛋白酶转染体之间的相对定量，从而验证了该技术的有效性，许多新的基质包括CX3、CL1趋化因子Fractalkine、骨桥蛋白、Galectin-1和HSP90α也被鉴定和生化证实。与ICAT标记和定量相比，iTRAQ鉴定的蛋白质和底物增加了8-9倍。通过iTRAQ确定特定蛋白质中多个多肽的位置，结合它们的相对丰度比，可以在天然细胞底物中确定结构域脱落和裂解位置的大致位置。因此，在Degradomics基于细胞的天然蛋白质底物筛选方面的这一进展为蛋白酶在细胞功能中的作用提供了新的线索。
Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2(-/-) fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQ(TM) mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this technique with many novel MMP-2 substrates including the CX3,CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90 alpha also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8-9-fold more proteins and substrates were identified by iTRAQ. "Peptide mapping," the location of multiple peptides identified within a particular protein by iTRAQ in combination with their relative abundance ratios, enabled the domain shed and general location of the cleavage site to be identified in the native cellular substrate. Hence this advance in degradomics cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.