Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A).
Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A).
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DOI：
10.3791/61138
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发表时间：
2020-06
期刊：
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通讯作者：
Y. Niikura;Lei Fang;R. Kitagawa;Peizhao Li;Y. Xi;Ju You;Yan Guo;K. Kitagawa
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文献类型：
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作者：
Y. Niikura;Lei Fang;R. Kitagawa;Peizhao Li;Y. Xi;Ju You;Yan Guo;K. Kitagawa
研究着丝粒和着丝粒的结构和动力学对于理解染色体不稳定性 (CIN) 和癌症进展非常重要。如何确定着丝粒的染色体位置和功能（即着丝粒身份）并参与准确的染色体分离是一个基本问题。 CENP-A被认为是着丝粒身份的非DNA指标（表观遗传标记），CENP-A泛素化是CENP-A在着丝粒沉积所必需的，通过细胞分裂之间的二聚化遗传，并且对于细胞活力是不可或缺的。在这里，我们描述了质谱分析来鉴定 EYFP-CENP-A K124R 突变体的泛素化，这表明由于 CENP-A K124R 突变体蛋白中的 EYFP 标签而诱导了不同赖氨酸的泛素化。通过质谱分析成功鉴定出EYFP-CENP-A K124R中的赖氨酸306（K306）泛素化，其对应于CENP-A中的赖氨酸56（K56）。讨论了使用 GFP/EYFP 或高分子量蛋白质标记作为分析蛋白质功能的工具的注意事项。还讨论了当前的技术限制，包括泛素化条带的检测、位点特异性泛素化的识别以及整个细胞周期中活细胞或特定单细胞中泛素化的可视化。本文提出的质谱分析方法可应用于具有不同标签的人类CENP-A蛋白和其他着丝粒着丝粒蛋白。这些由多种测定/分析组成的组合方法可以推荐给有兴趣识别泛素化功能作用的研究人员。
Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle. The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.