Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1105, Amsterdam 1081 HV, The Netherlands.
Centre for Analytical Sciences Amsterdam, Amsterdam 1098 XH, The Netherlands.
Anal Chem. 2024 Apr 2;96(13):5115-5124. doi: 10.1021/acs.analchem.3c04974. Epub 2024 Mar 22.
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-NH 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-NH, Ac-PHF6, PHF6-NH, 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.
肽和蛋白质的聚集涉及低聚物的形成，但不同构象和大小的低聚物之间的复杂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），我们旨在揭示来自 tau 蛋白的 Ac-PHF6-NH 肽段聚集的早期步骤，从而区分不同的低聚物物种并了解聚集途径。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，我们展示了使用 IM-MS 来探测 Ac-PHF6-NH、Ac-PHF6、PHF6-NH 和无封端 PHF6 肽段的聚集形成的早期阶段。使用硫黄素 T 荧光测定法和透射电子显微镜证实了这四个 PHF6 片段的聚集倾向。基于 TIMS-Qq-ToF（俘获离子淌度）光谱仪上的 IM 后碎片化和四极选择，开发了一种新方法，用于增强低聚物分配，特别是对于更高阶的聚集体。这种方法推动了等质异位体的 IM 鉴定的极限，随着低聚物尺寸的增加，其特征彼此之间更加接近，并为解释 IM-MS 数据提供了新的见解。此外，TIMS 碰撞截面值与行波离子淌度（TWIMS）数据进行比较，以评估在俘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和不同的配置，从而为我们提供了有价值的见解，了解对弱结合生物分子复合物（如肽聚集物）的保留情况。
