Identification of reduction-susceptible disulfide bonds in transferrin by differential alkylation using O(16)/O(18) labeled iodoacetic acid.
Identification of reduction-susceptible disulfide bonds in transferrin by differential alkylation using O(16)/O(18) labeled iodoacetic acid.
复制标题
使用 O(16)/O(18) 标记的碘乙酸通过差异烷基化鉴定转铁蛋白中易还原的二硫键。
DOI：
10.1007/s13361-015-1082-5
复制
发表时间：
2015
影响因子：
3.2
通讯作者：
Kaltashov,IgorA
中科院分区：
文献类型：
--
作者：
Wang,Shunhai;Kaltashov,IgorA
几十年来，稳定天然三维结构一直被认为是蛋白质中二硫键的主要功能。最近，人们越来越清楚，除了这种静态作用之外，二硫键对于蛋白质行为的许多其他方面也很重要，例如以氧化还原敏感的方式调节蛋白质功能。动态二硫键可用作位点特异性修饰的候选锚定位点（例如与药物分子缀合的聚乙二醇化），但也经常涉及蛋白质聚集（通过二硫键扰乱导致分子间共价连接的形成）。所有这些不稳定二硫键的一个共同特征是它们对还原高度敏感，因为它们需要通过体内特定的局部氧化还原条件或体外良好控制的实验条件进行选择性调节。识别富含半胱氨酸的蛋白质中不稳定的二硫键的能力对于各种任务都非常有益，从了解蛋白质功能的机制到识别生物制药产品中麻烦的“热点”。在此，我们描述了一种基于质谱 (MS) 的方法，用于可靠鉴定不稳定的二硫键，该方法包括有限还原、O18 标记试剂的差异烷基化以及 LC-MS/MS 分析。将该方法应用于富含半胱氨酸的蛋白转铁蛋白，可以测量其大部分天然二硫键的还原敏感性，这似乎反映了溶剂可及性和键应变能。
Stabilization of native three-dimensional structure has been considered for decades to be the main function of disulfide bonds in proteins. More recently, it was becoming increasingly clear that in addition to this static role, disulfide bonds are also important for many other aspects of protein behavior, such as regulating protein function in a redox-sensitive fashion. Dynamic disulfide bonds can be taken advantage of as candidate anchor sites for site-specific modification (such as PEGylation of conjugation to a drug molecule), but are also frequently implicated in protein aggregation (through disulfide bond scrambling leading to formation of intermolecular covalent linkages). A common feature of all these labile disulfide bonds is their high susceptibility to reduction, as they need to be selectively regulated by either specific local redox conditions in vivo or well-controlled experimental conditions in vitro. The ability to identify labile disulfide bonds in a cysteine-rich protein can be extremely beneficial for a variety of tasks ranging from understanding the mechanistic aspects of protein function to identification of troublesome “hot spots” in biopharmaceutical products. Herein, we describe a mass spectrometry (MS)-based method for reliable identification of labile disulfide bonds, which consists of limited reduction, differential alkylation with an O18-labeled reagent, and LC-MS/MS analysis. Application of this method to a cysteine-rich protein transferrin allows the majority of its native disulfide bonds to be measured for their reduction susceptibility, which appears to reflect both solvent accessibility and bond strain energy.