The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pK_a values of isoAsp and C-terminal Asp in short peptides. The characteristic ¹H-¹³C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

监测治疗性蛋白质中的非酶促翻译后修饰 (PTM) 对于确保药物安全性和有效性非常重要。与蛋氨酸和天冬酰胺一起，天冬氨酸 (Asp) 对自发变化非常敏感。特别是，Asp 残基会发生异构化和肽键水解，特别是当嵌入易于形成琥珀酰亚胺的序列基序中或后接脯氨酸 (Pro) 时。由于 Asp 和 isoAsp 具有相同的质量，并且 Asp-Pro 肽键裂解可能导致在天然条件下或在二硫键裂解产物的情况下出现 + 18 Da 的非特异性质量差异，因此直接检测和检测具有挑战性通过质谱 (MS) 表征这种修饰。在这里，我们提出了一种基于 2D NMR 的方法，用于明确鉴定 isoAsp 和 Asp-Pro 肽键裂解的产物，即 N 端 Pro 和 C 端 Asp，并证明其适用于包括治疗性单克隆抗体在内的蛋白质。单克隆抗体）。为了选择理想的 pH 条件，在该条件下 isoAsp 和 C 端 Asp 的 NMR 信号与其他随机线圈信号不同，我们确定了 pK_a短肽中 isoAsp 和 C 端 Asp_的值。标准化条件下 isoAsp、N 端 Pro 和 C 端 Asp的特征¹ H- ¹³ C 化学位移相关性用于鉴定溶菌酶和治疗性 mAb 利妥昔单抗 (MabThera) 在酸性条件 (pH 4–5) 和 40 °C。结果表明，我们基于 2D NMR 的协议的应用很简单，可以检测蛋白质的化学变化，否则其他分析方法可能会忽视这些变化。