Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

中文翻译：

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使用钠阳离子化试剂结合红外多光子解离通过气相离子/离子交联反应阐明泛素的结构


蛋白质的准确结构测定对于了解其生物学功能以及结构破坏对疾病进展的影响至关重要。气相交联质谱 (XL-MS) 通过多电荷蛋白质阳离子和单电荷交联剂阴离子之间的离子/离子反应，已被开发用于获取蛋白质的低分辨率结构信息。相对于传统的溶液相 XL-MS，该方法显着缩短了实验时间，但存在一些技术限制：(1) 单去质子化的 N-羟基磺基琥珀酰亚胺 (sulfo-NHS) 基交联剂阴离子仅限于连接在中性胺基上碱性氨基酸残基和（2）分析末端交联片段离子不足以明确定位接头附着位点。在此，我们展示了通过替代气相 XL-MS 方法获得的酒精变性 A 态泛素的增强结构信息。简而言之，单钠化乙二醇双（磺基琥珀酰亚胺基琥珀酸酯）（磺基-EGS）交联剂阴离子能够在铵基和胺基上实现共价交联。此外，共价修饰的内部碎片离子以及末端 b-/y-型对应物可改善接头附着位点的确定。分子动力学模拟验证了实验获得的变性泛素的气相构象。该方法已鉴定出 8+ 泛素的 4 个交联位点，其中包括蛋白质 N 末端区域的两个新位点，这两个位点在之前的气相 XL 方法中最初是无法接近的。两个 N 端交联位点表明泛素的 N 端一半在气相构象中更加紧凑。 通过比较，两个 C 端接头位点表明该蛋白质区域从天然构象到变性构象的标志性转变。总体而言，结果表明 A 态泛素的溶液相二级结构在气相中是保守的。该方法还提供足够的灵敏度来区分具有细微结构变化的相同电荷状态的两种气相构象异构体。