Peptide and protein aggregation involves the formation of oligomeric species, but the complex interplay between oligomers of different conformations and sizes complicates their structural elucidation. Using ion mobility mass spectrometry (IM-MS), we aim to reveal these early steps of aggregation for the Ac-PHF6-NH₂ peptide segment from tau protein, thereby distinguishing between different oligomeric species and gaining an understanding of the aggregation pathway. An important factor that is often neglected, but which can alter the aggregation propensity of peptides, is the terminal capping groups. Here, we demonstrate the use of IM-MS to probe the early stages of aggregate formation of Ac-PHF6-NH₂, Ac-PHF6, PHF6-NH₂, and uncapped PHF6 peptide segments. The aggregation propensity of the four PHF6 segments is confirmed using thioflavin T fluorescence assays and transmission electron microscopy. A novel approach based on post-IM fragmentation and quadrupole selection on the TIMS-Qq-ToF (trapped ion mobility) spectrometer was developed to enhance oligomer assignment, especially for the higher-order aggregates. This approach pushes the limits of IM identification of isobaric species, whose signatures appear closer to each other with increasing oligomer size, and provides new insights into the interpretation of IM-MS data. In addition, TIMS collision cross section values are compared with traveling wave ion mobility (TWIMS) data to evaluate potential instrumental bias in the trapped ion mobility results. The two IM-MS instrumental platforms are based on different ion mobility principles and have different configurations, thereby providing us with valuable insight into the preservation of weakly bound biomolecular complexes such as peptide aggregates.

中文翻译：

---

使用离子淌度质谱技术探索 Tau 蛋白 PHF6 肽段的聚集倾向

肽和蛋白质聚集涉及寡聚体的形成，但不同构象和大小的寡聚体之间复杂的相互作用使它们的结构阐明变得复杂。使用离子淌度质谱 (IM-MS)，我们的目的是揭示 tau 蛋白 Ac-PHF6-NH ₂肽段聚集的早期步骤，从而区分不同的寡聚体种类并了解聚集途径。一个经常被忽视但可以改变肽聚集倾向的重要因素是末端封端基团。在这里，我们演示了使用 IM-MS 来探测 Ac-PHF6-NH ₂、Ac-PHF6、PHF6-NH ₂和未加帽的 PHF6 肽片段聚集体形成的早期阶段。使用硫代黄素 T 荧光测定和透射电子显微镜证实了四个 PHF6 片段的聚集倾向。开发了一种基于 TIMS-Qq-ToF（俘获离子淌度）光谱仪上的 IM 后碎片和四极杆选择的新方法，以增强低聚物分配，特别是对于高阶聚集体。这种方法突破了同量异位物质的 IM 识别极限，随着寡聚物尺寸的增加，同量异位物质的特征显得彼此更接近，并为 IM-MS 数据的解释提供了新的见解。此外，还将 TIMS 碰撞截面值与行波离子淌度 (TWIMS) 数据进行比较，以评估捕获离子淌度结果中潜在的仪器偏差。这两个 IM-MS 仪器平台基于不同的离子淌度原理并具有不同的配置，从而为我们提供了保存弱结合生物分子复合物（例如肽聚集体）的宝贵见解。