Method for Quantifying Oxidized Methionines and Application to HIV-1 Env.
Method for Quantifying Oxidized Methionines and Application to HIV-1 Env.
复制标题
氧化蛋氨酸的定量方法及其在 HIV-1 环境中的应用。
DOI：
10.1007/s13361-018-2010-2
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发表时间：
2018
影响因子：
3.2
通讯作者：
Desaire,Heather
中科院分区：
文献类型：
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作者：
Shipman,JoshuaT;Go,EdenP;Desaire,Heather
重组表达的蛋白质在表达、纯化、储存和分析过程中容易被氧化；最容易被氧化的残留物是蛋氨酸。使用当前的定量分析方法可能会高估蛋氨酸的氧化，因为氧化可能在样品制备过程中发生，而研究人员通常不使用考虑这种可能性的方法。之前已经开发了一种实验策略来通过使用18 O标记的过氧化氢试剂来解决该问题。然而，该方法没有解决含有多个蛋氨酸残基的肽的分析。在此，我们开发并验证了一种新的分析方法，该方法使用理论同位素分布和实验光谱来量化样品制备之前存在的蛋氨酸氧化。新描述的方法比之前描述的方法更快，并且只需要一半的蛋白质量即可进行分析。该方法使用模型蛋白进行了验证；然后，它被应用于重组 HIV-1 Env（HIV 候选疫苗中的关键蛋白）的分析。虽然使用以前的方法无法分析该蛋白质的 Met 氧化，但本文描述的方法可用于确定 HIV-Env 的氧化态。
Recombinantly expressed proteins are susceptible to oxidation during expression, purification, storage, and analysis; the residue most susceptible to oxidation is methionine. Methionine oxidation can be overestimated using current quantitative analysis methods because oxidation can occur during sample preparation, and researchers often do not use methods that account for this possibility. An experimental strategy had been developed previously to solve this problem through the use of an18O-labeled hydrogen peroxide reagent. However, the method did not address the analysis of peptides that contained multiple methionine residues. Herein, we develop and validate a new analysis method that uses theoretical isotope distributions and experimental spectra to quantify methionine oxidation that is present prior to sample preparation. The newly described approach is more rapid than the previously described method, and it needs only half the amount of protein for analysis. This method was validated using model proteins; then, it was applied to the analysis of recombinant HIV-1 Env, the key protein in HIV vaccine candidates. While Met oxidation of this protein could not be analyzed using previous methods, the approach described herein was useful for determining the oxidation state of HIV-Env.