Edman Degradation Reveals Unequivocal Analysis of the Disulfide Connectivity in Peptides and Proteins,Analytical Chemistry

当前位置： X-MOL 学术 › Anal. Chem. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Edman Degradation Reveals Unequivocal Analysis of the Disulfide Connectivity in Peptides and Proteins
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-02-26 , DOI: 10.1021/acs.analchem.3c04229
Yomnah Y Elsayed _{1,

2} , Toni Kühl ₁ , Diana Imhof ₁

Affiliation

Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, μ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.

中文翻译：

Edman 降解揭示了肽和蛋白质中二硫键连接性的明确分析

肽和蛋白质中的二硫桥在维持其构象、结构完整性和功能方面发挥着重要作用。尽管不断努力，但仍然不可能通过简单且经过充分验证的蛋白质测序或肽图谱方法直接检测二硫键和多重桥肽的连接性。作为初始步骤，需要部分或完全还原和化学半胱氨酸修饰，然后应用适当的检测方法。埃德曼降解 (ED) 已被用于一级序列测定，但自从基于质谱 (MS) 的蛋白质测序建立以来，很大程度上被忽视了。在这里，我们评估并彻底表征了苯基硫代乙内酰脲 (PTH) 半胱氨酸衍生物 PTH-S-甲基半胱氨酸和 PTH-S-脲甲基半胱氨酸，作为半胱氨酸检测和定量以及通过 ED 阐明肽中二硫键连接性的生物分析标准品。根据国际协调委员会生物分析方法验证指南对所建立的衍生物进行验证，并确认其分析特性作为参考标准。对一系列模型肽进行测序，以测试 PTH-Cys 衍生物作为标准品的可用性，而天然二硫键肽 CCAP-vil、μ-芋螺毒素 KIIIA 和人胰岛素则用作案例研究，以确定其二硫键连接性完全独立于 MS 分析。

更新日期：2024-02-26

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南