Department of Bioengineering, Beijing Technology and Business University, Beijing, 100048, PR China.
National Engineering Research Center for Protein Drugs, Beijing C&N International Sci-tech Co., Ltd., Beijing, 102206, PR China.
Anal Bioanal Chem. 2024 Dec;416(29):6725-6733. doi: 10.1007/s00216-024-05597-y. Epub 2024 Oct 30.
Disulfide bridge, an important post-translation modification in protein, plays a key role in stabilizing three-dimensional structure of proteins, maintaining correct folded conformation, and thus regulating the biological activities. Disulfide bridge assignment is essential to understand the essence of life process and to develop protein pharmaceutical. In this study, a novel method termed as stepwise reduction and differentiated alkylation (SRDA) was developed analyzing disulfide connectivity for proteins. As a demonstration, three disulfide bridges in insulin aspart were successfully characterized using this SRDA method combined with LC-HRMS. Firstly, tris (2-carboxyethyl) phosphine (TCEP) was used to partially reduce disulfide bridges with N-ethylmaleimide (NEM) used to block the generated free thiol. Then, dithiothreitol (DTT) was used to reduce the rest disulfide bonds with iodoacetamide (IAM) used to block the newly generated free thiol. After that, an LC-HRMS method was established to assign disulfide connectivity for an insulin aspart study material, based on the different mass shifts arising from differentiated alkylation. Moreover, this approach allows for the quantitative analysis of various disulfide bond pairings, which can be applied to studies on the consistency and stability among different batches of samples. The results show that SRDA is a valuable tool for reliable quality control and quality assessment of disulfide-rich proteins such as insulin analogues.
二硫键是蛋白质翻译后的一种重要修饰，在稳定蛋白质的三维结构、维持正确的折叠构象以及调节生物活性方面发挥着关键作用。二硫键的确定对于理解生命过程的本质和开发蛋白质药物至关重要。在本研究中，开发了一种称为逐步还原和差异烷基化（SRDA）的新方法，用于分析蛋白质中二硫键的连接。作为演示，该方法结合 LC-HRMS 成功地对胰岛素Asp 中的三个二硫键进行了特征分析。首先，使用三（2-羧乙基）膦（TCEP）部分还原二硫键，并用 N-乙基马来酰亚胺（NEM）封闭生成的游离巯基。然后，用二硫苏糖醇（DTT）还原其余的二硫键，并用碘乙酰胺（IAM）封闭新生成的游离巯基。之后，基于差异烷基化产生的不同质量位移，建立了用于胰岛素 Asp 研究材料中二硫键连接的 LC-HRMS 方法。此外，该方法还可以定量分析各种二硫键配对，适用于不同批次样品之间一致性和稳定性的研究。结果表明，SRDA 是一种可靠的质量控制和质量评估工具，可用于二硫键丰富的蛋白质（如胰岛素类似物）。
