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 an ¹⁸O-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.

Open image in new window

重组表达的蛋白质在表达，纯化，储存和分析过程中易于氧化；最容易氧化的残基是蛋氨酸。使用蛋氨酸的氧化可以使用当前的定量分析方法高估，因为氧化可能会在样品制备过程中发生，并且研究人员通常不会使用能说明这种可能性的方法。先前已经开发出一种实验策略，通过使用¹⁸O标记的过氧化氢试剂。但是，该方法没有解决包含多个蛋氨酸残基的多肽的分析问题。在这里，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。新描述的方法比先前描述的方法更快，并且仅需要一半的蛋白质量即可进行分析。该方法已通过模型蛋白验证。然后，将其用于分析HIV候选疫苗中的关键蛋白重组HIV-1 Env。尽管无法使用以前的方法分析该蛋白质的Met氧化，但本文所述方法可用于确定HIV-Env的氧化状态。

在新窗口中打开图像