Identification of disulfide-containmg chemical cross links in proteins using MALDI-TOF/TOF-mass spectrometry
Identification of disulfide-containmg chemical cross links in proteins using MALDI-TOF/TOF-mass spectrometry
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DOI：
10.1021/ac702277q
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发表时间：
2008-07-01
影响因子：
7.4
通讯作者：
Ross, Ian L.
中科院分区：
文献类型：
--
作者：
King, Gordon J.;Jones, Alun;Ross, Ian L.
交联可用于鉴定蛋白质或蛋白质复合物中氨基酸之间的空间关系。提出了一种快速、灵敏的方法，用于鉴定使用二硫代双（磺基琥珀酰亚胺基丙酸酯）（DTSSP）的蛋白质交联的位点，并用鼠皮质素、肌动蛋白和酰基辅酶A硫酯酶的实验进行了说明。在MALDI-TOF/TOF-MS条件下获得的质谱中观察到一个特征性的66 Da双峰，其来自DTSSP修饰肽的二硫化物的不对称断裂，并允许快速分配修饰蛋白质中的交联。这种双重峰不仅在线性交联肽中观察到，而且在环状交联肽的质谱中也观察到，当二硫化物和肽骨架同时发生断裂时。我们提出了一个可能的机制，这种分裂。我们使用胍基化的交联肽与O-甲基异硫氰酸酯，以扩大在此MALDI-MS技术中观察到的交联肽的覆盖范围。我们报告的方法是强大的，适合自动化，并允许分析天然胱氨酸沿着与那些引入含二硫化物的交联剂。
Cross-linking can be used to identify spatial relationships between amino acids in proteins or protein complexes. A rapid and sensitive method for identifying the site of protein cross-linking using dithiobis(sulfosuccinimidyl propionate) (DTSSP) is presented and illustrated with experiments using murine cortactin, actin and acyl-CoA thioesterase. A characteristic 66 Da doublet, which arises from the asymmetric fragmentation of the disulfide of DTSSP-modified peptides, is observed in the mass spectra obtained under MALDI-TOF/TOF-MS conditions and allows rapid assignment of cross-links in modified proteins. This doublet is observed not only for linear cross-linked peptides but also in the mass spectra of cyclic cross-linked peptides when simultaneous fragmentation of the disulfide and the peptide backbone occurs. We suggest a likely mechanism for this fragmentation. We use guanidinylation of the cross-linked peptides with O-methyl isourea to extend the coverage of cross-linked peptides observed in this MALDI-MS technique. The methodology we report is robust and amenable to automation, and permits the analysis of native cystines along with those introduced by disulfide-containing cross-linkers.