Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
J Am Chem Soc. 2024 Oct 23;146(42):28809-28821. doi: 10.1021/jacs.4c08455. Epub 2024 Oct 10.
Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity () of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.
蛋白质寡聚化调控着许多关键的生理过程，其失调可能导致功能障碍和疾病。阐明组装途径并量化其潜在的热力学和动力学参数，对于全面理解生物学过程和推进针对异常蛋白质寡聚化的治疗方法至关重要。已建立的结合测定法，其质量精度有限，通常依赖于简化的模型进行数据解释。相比之下，高分辨率的天然质谱（nMS）可以直接确定体外生物分子复合物的化学计量。然而，由于气相离子的相对丰度通常不能反映溶液浓度，因为响应因子不均匀，所以定量受到阻碍。最近，慢混合模式（SLOMO）-nMS 已被证明可以可靠地测量二元生物分子复合物的亲和力（），该方法可以定量相互作用物质的相对响应因子。在这里，我们引入了一种扩展形式的 SLOMO-nMS，它可以同时定量多步缔合反应的热力学。该方法在伴刀豆球蛋白 A 和胰岛素的同寡聚化中的应用证实了该测定的可靠性，并揭示了以前难以阐明的组装过程的细节。使用带有电荷检测的 SLOMO-nMS 获得的结果为重组人血管紧张素转换酶 2 和 SARS-CoV-2 刺突蛋白的结合提供了新的见解。重要的是，发现了新的组装途径，并对这些调节宿主细胞感染的相互作用的亲和力进行了量化。总之，这些发现突显了 SLOMO-nMS 极大地加速了蛋白质组装途径和热力学的表征的潜力，并以此增强了基本的生物学理解，促进了治疗方法的发展。
