Department of Proteomics, The NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.
Mol Cell Proteomics. 2012 Dec;11(12):1578-85. doi: 10.1074/mcp.M112.017905. Epub 2012 Jul 11.
Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.
蛋白质的翻译后修饰增加了细胞蛋白质组的复杂性，并能够快速调节蛋白质的功能以响应环境变化。蛋白质泛素化是一种核心的调节翻译后修饰，控制着许多生物学过程，包括蛋白质的蛋白酶体降解、DNA 损伤修复和先天免疫反应。在这里，我们将高分辨率质谱与二甘氨酸修饰肽的单步免疫富集相结合，用于绘制鼠组织中内源性假定泛素化位点的图谱。我们在涉及多种生物学过程的蛋白质上鉴定了超过 20000 个独特的泛素化位点。我们的数据表明，泛素化调节了每个研究组织共有的核心信号通路。此外，我们发现泛素化调节了组织特异性的信号网络。许多组织特异性的泛素化位点来自于大脑，突出了该器官的复杂性和独特的生理学。我们进一步证明，不同的二甘氨酸-赖氨酸特异性单克隆抗体表现出序列偏好，并且它们的互补使用增加了泛素化位点分析的深度，从而提供了对蛋白质泛素化的更客观的看法。
