Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry
Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry
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DOI：
10.1002/pmic.200600410
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发表时间：
2007-03-01
期刊：
影响因子：
3.4
通讯作者：
Figeys, Daniel
中科院分区：
文献类型：
--
作者：
Denis, Nicholas J.;Vasilescu, Julian;Figeys, Daniel
泛素化通过调节许多重要过程在维持细胞内稳态中起着重要作用。通过MS鉴定泛素化蛋白的能力目前依赖于这样的策略，其中泛素化肽通过赖氨酸残基上的114.1 Da双甘氨酸（GG）标签鉴定，所述赖氨酸残基衍生自泛素的C-末端，在胰蛋白酶消化后。在接下来的研究中，我们报告了一个更全面的方法定位泛素化位点的胰蛋白酶消化和MS/MS分析。我们证明了泛素化。位点可以通过在内部赖氨酸残基上含有GG标签（114.1 Da）和LRGG标签（383.2 Da）以及在泛蛋白化肽的C末端发现的GG标签的特征肽来识别。这种基于MS的方法的应用使得能够从从人MCF-7乳腺癌细胞纯化的蛋白质中鉴定96个泛素化位点，这代表了比标准方法可鉴定的泛素化位点数量增加2.4倍。我们改进的基于MS的策略将有助于未来的研究，旨在识别和/或表征人类细胞中的泛素化蛋白。
Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination. sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.