Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
Institute of Organic Chemistry, University of Innsbruck , A-6020 Innsbruck, Austria.
Anal Chem. 2017 Oct 17;89(20):10711-10716. doi: 10.1021/acs.analchem.7b01581. Epub 2017 Oct 4.
Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.
天然电子捕获解离（NECD）是一种蛋白质发生碎裂的过程，其碎裂方式类似于自由基解离方法，但无需外加外源电子。然而，在最初有三篇关于细胞色素 c 二聚体复合物的 NECD 的报道之后，再没有发表过关于该效应的进一步证据。在这里，我们报道了马脾铁蛋白的 NECD 行为，马脾铁蛋白是一种 ∼490 kDa 的蛋白质复合物，比之前研究的细胞色素 c 二聚体大 20 倍。前端红外激发（FIRE）与低质荷比和高质荷比四极杆隔离和碰撞激活解离（CAD）的联合应用为 NECD 机制提供了新的见解。此外，在电喷雾液滴或气相中激活完整复合物会产生 c 型片段离子。与之前报道的细胞色素 c 的结果类似，这些片段离子在与溶液中铁原子相互作用的残基附近形成。通过将与 c 型离子相关的骨架裂解的位置映射到晶体结构上，我们能够表征两个不同的铁结合通道，这些通道有利于铁离子进入复合物核心。得到的途径与哺乳动物铁蛋白中铁结合位点的先前报道结果非常吻合。
