Department of Mechanical and Information Engineering, University of Seoul, Seoul, Korea.
J Proteome Res. 2010 Jan;9(1):626-35. doi: 10.1021/pr900771r.
Identifying the sites of disulfide bonds in a protein is essential for thorough understanding of a protein's tertiary and quaternary structures and its biological functions. Disulfide linked peptides are usually identified indirectly by labeling free sulfhydryl groups with alkylating agents, followed by chemical reduction and mass spectral comparison or by detecting the expected masses of disulfide linked peptides on mass scan level. However, these approaches for determination of disulfide bonds become ambiguous when the protein is highly bridged and modified. For accurate identification of disulfide linked peptides, we present here an algorithmic solution for the analysis of tandem mass (MS/MS) spectra of disulfide bonded peptides under nonreducing condition. A new algorithm called "DBond" analyzes disulfide linked peptides based on specific features of disulfide bonds. To determine disulfide linked sites, DBond takes into account fragmentation patterns of disulfide linked peptides in nucleoside diphosphate kinase (NDPK) as a model protein, considering fragment ions including cysteine, cysteine thioaldehyde (-2 Da, C(T)), cysteine persulfide (+32 Da, C(S)) and dehydroalanine (-34 Da, C(Delta)). Using this algorithm, we successfully identified about a dozen novel disulfide bonds in a hexa EF-hand calcium binding protein secretagogin and in a methionine sulfoxide reductase. We believe that DBond, taking into account the disulfide bond fragmentation characteristics and post-translational modifications, offers a novel approach for automatic identification of unknown disulfide bonds and their sites in proteins from MS/MS spectra.
鉴定蛋白质中二硫键的位置对于深入了解蛋白质的三级和四级结构及其生物功能至关重要。二硫键连接的肽段通常通过用烷基化试剂标记游离巯基基团，然后进行化学还原和质谱比较，或者在质谱扫描水平上检测二硫键连接肽段的预期质量来间接鉴定。然而，当蛋白质高度桥接和修饰时，这些确定二硫键的方法变得不确定。为了准确鉴定二硫键连接的肽段，我们在这里提出了一种在非还原条件下分析二硫键连接肽段串联质谱（MS/MS）谱的算法解决方案。一种名为“DBond”的新算法基于二硫键的特定特征来分析二硫键连接的肽段。为了确定二硫键连接的位点，DBond 考虑了核昔二磷酸激酶（NDPK）作为模型蛋白中二硫键连接肽段的断裂模式，同时考虑了包括半胱氨酸、半胱氨酸硫醛（-2 Da，C(T)）、半胱氨酸过硫化物（+32 Da，C(S)）和脱氢丙氨酸（-34 Da，C(Delta)）在内的片段离子。使用该算法，我们成功地鉴定了六 EF 手钙离子结合蛋白 secretagogin 和甲硫氨酸亚砜还原酶中的十几个新的二硫键。我们相信，DBond 考虑了二硫键断裂特征和翻译后修饰，为从 MS/MS 谱中自动鉴定蛋白质中的未知二硫键及其位点提供了一种新方法。
