Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC–MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

中文翻译：

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使用 2-亚甲基丙二酸二乙酯对硫醇进行酸性衍生：硫醇-迈克尔加成点击反应，使用 LC-MS/MS 同时分析培养基中的半胱氨酸和胱氨酸

半胱氨酸 (Cys) 及其氧化形式胱氨酸 (Cys2) 在生物系统中发挥着至关重要的作用，并且在细胞培养中具有广泛的应用。然而，细胞培养基中的Cys很容易被氧化成Cys2，导致溶解度问题。传统的分析方法在分析过程中难以维持 Cys 和 Cys2 的氧化态，这对准确测量和控制这些化合物提出了重大挑战。为了有效控制Cys和Cys2水平，需要快速、准确的分析方法。在这里，我们筛选了即使在酸性条件下也能与Cys反应的衍生试剂，以实现一种同时测定Cys和Cys2水平的新分析方法。 2-亚甲基丙二酸二乙酯 (EMM) 被发现具有所需的特性。 EMM 具有双吸电子属性，可在酸性条件下与 Cys 快速反应，保留完整的信息以了解目标化合物的功能。该方法与 LC-MS/MS 和内标相结合，可在 9 分钟的短时间内提供较高的分析精度。使用所开发的方法，观察到细胞培养基中 Cys 的快速氧化，储存容器的顶部空间显着影响 Cys 氧化和 Cys2 沉淀速率。所开发的方法能够直接、简化地分析实际培养基样品中的 Cys 行为，并可用于配制新的培养基组合物、确保质量保证以及细胞培养上清液中 Cys 和 Cys2 的实时分析。这种新颖的方法有可能通过及时优化添加半胱氨酸来进一步提高介质性能。