Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

译文

:非常接近的氨基酸残基之间的交联可为3D结构蛋白模型的验证提供距离限制。通过可裂解的交联剂促进了通过蛋白水解酶消化物中的连接肽的鉴定来进行交联的作图,所述可裂解的交联剂能够分离裂解产物,同时保留有关连锁的信息。我们提出了满足这些要求的特定于胺的交联剂,双(琥珀酰亚胺基)-3-叠氮基甲基戊二酸酯(BAMG)。三(羧乙基)膦(TCEP)在来自BAMG处理的细胞色素c的交联肽中诱导出两个平行的反应途径。一个途径导致交联物种的裂解,而在另一途径中,BAMG的叠氮基还原为氨基而不裂解。交联的肽和经部分水解的BAMG修饰的肽可产生不同组的TCEP诱导的反应产物。这些可以通过反相对角色谱法分离,并通过质谱鉴定以揭示母体化合物的身份。易于分离和鉴定交联衍生的反应产物表明,在不久的将来,复杂生物组装体和蛋白质复合物混合物中交联的作图可能变得可行。

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