Deamidation of asparagine is a spontaneous and irreversible post-translational modification associated with a growing list of human diseases. While pervasive, deamidation is often overlooked because it represents a relatively minor chemical change. Structural and functional characterization of this modification is complicated because deamidation of asparagine yields four isomeric forms of Asp. Herein, radical directed dissociation (RDD), in conjunction with mass spectrometry, is used to identify and quantify all four isomers in a series of model peptides that were subjected to various deamidation conditions. Although primary sequence significantly influences the rate of deamidation, it has little impact on the relative proportions of the product isomers. Furthermore, the addition of ammonia can be used to increase the rate of deamidation without significantly perturbing isomer populations. Conversely, external factors such as buffer conditions and temperature alter product distributions but exhibit less dramatic effects on the deamidation rate. Strikingly, the common laboratory and biologically significant bicarbonate buffer is found to strongly promote racemization, yielding increased amounts of d-Asp and d-isoAsp. These outcomes following deamidation have broad implications in human aging and should be considered during the development of protein-based therapeutics.

中文翻译：

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序列和溶液对脱酰胺作用d-异构体发生率的影响

天冬酰胺的脱酰胺作用是自发且不可逆的翻译后修饰，与越来越多的人类疾病有关。尽管脱酰胺作用普遍存在，但由于它代表相对较小的化学变化，因此常常被忽略。该修饰的结构和功能表征很复杂，因为天冬酰胺的脱酰胺作用会产生4种Asp异构形式。在本文中，自由基定向解离（RDD）与质谱法一起用于鉴定和量化遭受各种脱酰胺条件的一系列模型肽中的所有四个异构体。尽管一级序列显着影响脱酰胺速率，但对产物异构体的相对比例影响很小。此外，氨的添加可用于增加脱酰胺速率，而不会显着干扰异构体的数量。相反，诸如缓冲液条件和温度之类的外部因素会改变产物的分布，但对脱酰胺速率的影响较小。令人惊讶的是，发现普通的实验室和生物学上重要的碳酸氢盐缓冲液可强烈促进外消旋作用，从而产生大量的外消旋体。d -Asp和d -isoAsp。脱酰胺后的这些结果对人类衰老具有广泛的意义，应在开发基于蛋白质的疗法时加以考虑。