UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites
UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites
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DOI：
10.1038/s41594-018-0084-y
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发表时间：
2018-07-01
影响因子：
16.8
通讯作者：
Blagoev, Blagoy
中科院分区：
文献类型：
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作者：
Akimov, Vyacheslav;Barrio-Hernandez, Inigo;Blagoev, Blagoy
泛素化是一种翻译后修饰（PTM），对于平衡许多生理过程至关重要。为了能够在位点特异性水平上描绘蛋白质泛素化，我们产生了一种抗体，称为UbiSite，识别泛素的C-末端13个氨基酸，其在用内切蛋白酶LysC进行蛋白水解消化后仍然附着在修饰的肽上。值得注意的是，UbiSite对泛素是特异性的。此外，除了赖氨酸残基上的泛素化，蛋白质N-末端泛素化也容易检测到。通过将UbiSite富集与连续LysC和胰蛋白酶消化以及高精度MS相结合，我们在两种人类细胞系中的9，200种蛋白质上鉴定了超过63，000个独特的泛素化位点。除了揭示广泛参与此PTM在所有细胞方面，分析揭示了蛋白质N-末端泛素化和乙酰化之间的负相关，以及蛋白质丰度的变化和蛋白酶体抑制后泛素化位点的改变之间完全缺乏相关性。
Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.