Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Nat Struct Mol Biol. 2018 Jul;25(7):631-640. doi: 10.1038/s41594-018-0084-y. Epub 2018 Jul 2.
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.
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。为了能够在特定的位点上对蛋白质的泛素化进行描绘，我们生成了一种抗体，称为 UbiSite，它可以识别泛素的 C 末端 13 个氨基酸残基，这些残基在 LysC 内切蛋白酶消化后仍然附着在修饰的肽段上。值得注意的是，UbiSite 是特异性针对泛素的。此外，除了赖氨酸残基上的泛素化之外，蛋白质 N 末端的泛素化也很容易被检测到。通过将 UbiSite 富集与连续的 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鉴定出了 9200 种蛋白质上超过 63000 个独特的泛素化位点。除了揭示这种 PTM 在所有细胞方面的广泛参与外，分析还揭示了蛋白质 N 末端泛素化与乙酰化之间的反比关系，以及蛋白酶体抑制后蛋白质丰度的变化与泛素化位点的改变之间完全没有相关性。
