Laboratories of Analytical Chemistry and Biochemistry, and Graduate School, Chemical Biology, University of Konstanz, Konstanz, Germany.
J Am Soc Mass Spectrom. 2011 Aug;22(8):1463-71. doi: 10.1007/s13361-011-0158-0. Epub 2011 May 24.
Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS-MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS-MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.
泛素是一种由 76 个氨基酸组成的关键细胞调节多肽，其赖氨酸残基之一通过赖氨酸特异性异肽键与多泛素缀合，这已被认为是决定其生化特性和细胞功能的主要途径。然而，不同赖氨酸异肽基泛素缀合物的结构细节和差异，反映了它们不同的功能和反应性，仅部分被理解。离子淌度谱（IMS）与质谱（MS）联用，最近已成为一种强大的工具，通过电场驱动在漂移气体中分离多肽离子，从而探测涉及蛋白质相互作用的构象和拓扑结构。在这里，我们报告了 IMS-MS 对 Lys63 和 Lys48 连接的泛素缀合物的构象特征和区分。Lys63 和 Lys48 连接的二泛素缀合物通过重组细菌表达和使用特定的化学连接策略进行化学合成来制备，并通过高分辨率傅里叶变换离子回旋共振质谱、圆二色光谱和分子建模进行了表征。发现 IMS-MS 是鉴定气相中泛素复合物结构差异的有效工具。Lys63 和 Lys48 连接的二泛素缀合物的碰撞截面比较表明，Lys63 连接的二泛素具有更拉长的构象。相比之下，Lys48 连接的二泛素缀合物呈现出更紧凑的构象。IMS-MS 结果与已发表的结构数据以及 Lys63 和 Lys48 连接的缀合物的比较分子建模研究一致。这里呈现的结果表明，IMS 技术可以提供补充 MS 测量的信息，用于区分高阶多泛素和其他异构蛋白质键。
