Riggs Dylan L, Gomez Sonia V, Julian Ryan R
Department of Chemistry, University of California , Riverside, California 92521, United States.
ACS Chem Biol. 2017 Nov 17;12(11):2875-2882. doi: 10.1021/acschembio.7b00686. Epub 2017 Oct 23.
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.
天冬酰胺的脱酰胺作用是一种自发且不可逆的翻译后修饰，与越来越多的人类疾病相关。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，常常被忽视。这种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四种天冬氨酸的异构体形式。在此，自由基导向解离（RDD）与质谱联用，用于鉴定和定量一系列在各种脱酰胺条件下的模型肽中的所有四种异构体。虽然一级序列显著影响脱酰胺速率，但对产物异构体的相对比例影响很小。此外，添加氨可用于提高脱酰胺速率，而不会显著扰乱异构体群体。相反，诸如缓冲条件和温度等外部因素会改变产物分布，但对脱酰胺速率的影响较小。引人注目的是，发现常见的实验室和生物学上重要的碳酸氢盐缓冲液会强烈促进消旋作用，产生更多的d-天冬氨酸和d-异天冬氨酸。脱酰胺作用后的这些结果对人类衰老具有广泛影响，在基于蛋白质的治疗药物开发过程中应予以考虑。
