Department of Chemistry, University of California-Riverside, 900 University Ave., Riverside, CA, 92521, USA.
Department of Environmental Toxicology Program, University of California-Riverside, 00 University Ave., Riverside, CA, 92521, USA.
Anal Bioanal Chem. 2020 Sep;412(24):6189-6198. doi: 10.1007/s00216-020-02486-y. Epub 2020 Feb 17.
Post-translational modifications (PTMs) greatly increase protein diversity and regulate their functions by changing the structures, properties, and molecular interactions of proteins. In peptide regions with high density of PTMs, PTMs can influence modification on residues in proximity or even at distal positions, adding another layer of regulation. Methods that can monitor the activities of PTM enzymes on peptides carrying multiple modifications are valuable tools for better understanding of PTM crosstalk. Herein, we developed a host-assisted capillary electrophoresis (CE) method to separate histone peptides with methylation and phosphorylation and applied it to monitor the crosstalk between serine phosphorylation and lysine methylation when they were added by Aurora B kinase and G9a lysine methyltransferase, respectively. A synthetic receptor molecule, 4-hexasulfonatocalix[6]arene (CX6), was included in the CE buffer to improve the resolution of the corresponding substrates and products. A linear polyacrylamide-coated capillary was employed to effectively reduce wall adsorption of the cationic histone peptides. The peptide substrates were labeled with fluorescein to enhance their detectability during CE separation. Our method successfully revealed that the activity of G9a methyltransferase was completely inhibited by the adjacent phosphorylation, while 25% reduction in the activity of Aurora B kinase was observed with the presence of dimethylation on the nearby residue. The PTM crosstalk was examined not only using a pure peptide substrate, but also in a competitive reaction environment, in which the modified and unmodified peptides were mixed and the enzyme actions on both peptides were monitored simultaneously. Our work demonstrates that host-assisted CE is an effective method for study of PTM crosstalk, which could offer the advantages of fast separation, high resolution, and low sample consumption. Graphical abstract.
翻译后修饰（PTMs）极大地增加了蛋白质的多样性，并通过改变蛋白质的结构、性质和分子相互作用来调节其功能。在具有高密度PTMs的肽区域中，PTMs可以影响邻近甚至远端位置残基的修饰，增加了另一层调控。能够监测PTM酶对携带多种修饰的肽的活性的方法，是更好地理解PTM串扰的有价值工具。在此，我们开发了一种宿主辅助毛细管电泳（CE）方法，用于分离具有甲基化和磷酸化的组蛋白肽，并将其应用于监测分别由极光激酶B和G9a赖氨酸甲基转移酶添加的丝氨酸磷酸化和赖氨酸甲基化之间的串扰。CE缓冲液中包含一种合成受体分子4-六磺酸杯[6]芳烃（CX6），以提高相应底物和产物的分离度。使用线性聚丙烯酰胺涂层毛细管有效地减少阳离子组蛋白肽的壁吸附。肽底物用荧光素标记，以增强其在CE分离过程中的可检测性。我们的方法成功地揭示了相邻的磷酸化完全抑制了G9a甲基转移酶的活性，而在附近残基存在二甲基化的情况下，观察到极光激酶B的活性降低了25%。不仅使用纯肽底物，而且在竞争反应环境中检查PTM串扰，在竞争反应环境中，将修饰和未修饰的肽混合，并同时监测两种肽上的酶作用。我们的工作表明，宿主辅助CE是研究PTM串扰的有效方法，它具有分离速度快、分辨率高和样品消耗低的优点。图形摘要。
