Barnett Institute of Chemical and Biological Analysis and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
J Am Soc Mass Spectrom. 2013 Jan;24(1):125-33. doi: 10.1007/s13361-012-0510-z. Epub 2012 Dec 4.
Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.
尽管当前在肽图分析方面取得了技术进步，采用高分辨率液相色谱与质谱联用（LC-MS），但半胱氨酸环或嵌套二硫键在结构上仍难以表征。在重组人芳基硫酸酯酶 A（rhASA）的情况下，C 末端有一个半胱氨酸环，中间有一对嵌套二硫键，N 末端区域有三个不成对半胱氨酸中的两个。这些半胱氨酸的状态是 rhASA 功能和稳定性的关键结构属性，需要进行精确检查。我们采用了一种独特的方法来确定 rhASA 中每个半胱氨酸的状态和连接，该方法包括多酶消化策略（来自 Lys-C、胰蛋白酶、Asp-N、胃蛋白酶和 PNGase F）和质谱中的多片段化方法，使用电子转移解离（ETD）、碰撞诱导解离（CID）和 CID 与 MS(3)（ETD 后）。除了生成用于有效片段化的所需长度的酶肽外，还优化了消化 pH 值以最大程度减少二硫键重排。二硫键连接包括半胱氨酸环和一对嵌套半胱氨酸、不成对半胱氨酸以及半胱氨酸形成甲酰基甘氨酸的翻译后修饰，所有这些都已确定。在分配中，二硫键连接为 Cys138-Cys154、Cys143-Cys150、Cys282-Cys396、Cys470-Cys482、Cys471-Cys484 和 Cys475-Cys481。对于不成对的半胱氨酸，Cys20 和 Cys276 是游离半胱氨酸，而 Cys51 则主要转化为甲酰基甘氨酸（>70%）。已经开发出一种成功的方法，可以常规用于确定这些难以解决的二硫键连接，以确保药物的功能和稳定性。
