BACKGROUND Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex arrangements (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as posttranslational modifications (PTMs). PTMs may affect the protein's functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins. METHODS In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones. RESULTS Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs. CONCLUSION This work required some novel computational approaches to pre-process Raman and IR spectra and a review of the band assignments for proteins existing in the literature.

中文翻译：

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探索分子光谱在检测受压生物药物蛋白的翻译后修饰方面的潜力。

背景技术蛋白质是由氨基酸序列（一级结构）组成的生物分子，这些氨基酸序列可以进一步相互作用并导致骨架折叠成更复杂的排列（二级和三级结构）。将信使 RNA 代码翻译成蛋白质一级序列后，分子的任何化学变化都称为翻译后修饰 (PTM)。PTM 可能会影响蛋白质的功能；因此有必要识别它们。制药行业特别感兴趣的 PTM 包括脱酰胺、氧化、去糖基化和异构化，这些可能由于环境压力而发生。然而，事实证明，它们很难快速识别。电子和振动光谱已被证明是研究蛋白质高阶结构和稳定性的有价值的工具。方法 在这项工作中，应用圆二色性 (CD)、红外吸光度 (IR) 和拉曼光谱来表征不同应激源导致的抗体 (mAb NIP 228) PTM。质谱法用于确认修饰的身份，包括目标修饰。结果室温CD显示所有处理后二级结构相同，温控CD显示蛋白质稳定性如何受到修饰的影响。拉曼和 IR 分析都检测到参考蛋白和去糖基化蛋白之间的微小差异，并清楚地表明存在其他 PTM。结论 这项工作需要一些新的计算方法来预处理拉曼和红外光谱，并审查文献中存在的蛋白质的谱带分配。应用红外吸光度 (IR) 和拉曼光谱来表征不同应激源导致的抗体 (mAb NIP 228) PTM。质谱法用于确认修饰的身份，包括目标修饰。结果室温CD显示所有处理后二级结构相同，温控CD显示蛋白质稳定性如何受到修饰的影响。拉曼和 IR 分析都检测到参考蛋白和去糖基化蛋白之间的微小差异，并清楚地表明存在其他 PTM。结论 这项工作需要一些新的计算方法来预处理拉曼和红外光谱，并审查文献中存在的蛋白质的谱带分配。应用红外吸光度 (IR) 和拉曼光谱来表征不同应激源导致的抗体 (mAb NIP 228) PTM。质谱法用于确认修饰的身份，包括目标修饰。结果室温CD显示所有处理后二级结构相同，温控CD显示蛋白质稳定性如何受到修饰的影响。拉曼和 IR 分析都检测到参考蛋白和去糖基化蛋白之间的微小差异，并清楚地表明存在其他 PTM。结论 这项工作需要一些新的计算方法来预处理拉曼和红外光谱，并审查文献中存在的蛋白质的谱带分配。