Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.
Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.
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Ac-LysargiNase 补充胰蛋白酶以鉴定泛素化位点。
DOI：
10.1021/acs.analchem.9b04340
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发表时间：
2019
影响因子：
7.4
通讯作者：
Xu Ping
中科院分区：
文献类型：
--
作者：
Xiao Weidi;Zhang Junling;Wang Yihao;Liu Zijuan;Wang Fuqiang;Sun Jinshuai;Chang Lei;Xia Zongping;Li Yanchang;Xu Ping
基于质谱（MS）的泛素化位点的鉴定需要在MS分析之前进行胰蛋白酶消化，并且产生具有连接到泛素化赖氨酸（K-ε-GG肽）的双甘氨酸残基的特征肽。然而，胰蛋白酶对修饰的赖氨酸的错过切割导致具有增加的长度和电荷的修饰的肽，其通过MS分析的检测被绝大多数内部未修饰的肽抑制。LysargiNase是镜像胰蛋白酶，据报道在赖氨酸和精氨酸残基之前切割，并且有利于甲基化和磷酸化的鉴定，但其与泛素化相关的消化特性尚不清楚。在此，我们测试了内部开发的具有高活性和稳定性的乙酰化LysargiNase（Ac-LysargiNase）切割七种类型的泛素链及其相应的K-ε-GG肽中的泛素化位点的能力。有趣的是，Ac-LysargiNase可以有效地切割K63连接的链，但对其他类型的链几乎没有影响。此外，Ac-LysargiNase具有比胰蛋白酶更高的外肽酶活性。利用配对镜像蛋白酶的这些特征，开发了胰蛋白酶和Ac-LysargiNase串联消化的工作流程用于鉴定泛素化蛋白。通过这种方法，有效地降低了未修饰肽的电荷状态和电离能力，从而使修饰位点的识别率提高了30%~ 50%。引人注目的是，大约15%的修饰位点被Ac-LysargiNase切割，导致更短的K-ε-GG肽用于更好的鉴定。Ac-LysargiNase酶有望成为提高泛素研究中修饰位点识别效率的一种选择。
Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.