Institute of Neuroimmunology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 10 Bratislava, Slovak Republic.
Axon Neuroscience R&D Services SE, Dvořákovo nábrežie 10, 811 02 Bratislava, Slovak Republic.
J Am Soc Mass Spectrom. 2023 Mar 1;34(3):394-400. doi: 10.1021/jasms.2c00289. Epub 2023 Jan 27.
Alzheimer's disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility-mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau-TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions (/ 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at / 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.
阿尔茨海默病（AD）是一种日益受到关注的神经退行性疾病。它属于被称为tau 病的疾病，其特征是tau 蛋白的异常过度磷酸化和截断形式的包含物。tau 病的研究通常集中于这些异常tau 蛋白形式的检测和特征描述，特别是磷酸化位点，这对分析来说是一个重大挑战，例如，在同一肽上可能存在多个磷酸化位点。这些异构体甚至可能难以完全通过色谱法完全分离。由于最近引入的环状离子迁移-质谱法可以提供不同的选择性，我们研究了来自富含脯氨酸的 tau-TPSLPTPPTREPK 区域的胰蛋白酶肽的紧密定位的磷酸化位点 S214、T212 和 T217。由于重叠的到达时间分布，气相中异构肽的构象异质性阻碍了它们的分离。仅增加分析的分辨率不足以区分患者样本中典型混合物中的肽。因此，我们开发了一种基于碰撞诱导解离、异构产物离子（/ 677）迁移率分离和迁移后解离相结合的方法，以帮助分析疾病脑组织提取物中 tau 的异构磷酸肽。对于所有三种异构体（T212、S214 和 T217），在 0.1 nmol/L 的浓度下，仍可以观察到离子在 / 677 的离子迁移信号。这项工作不仅深入了解了 AD 中 tau 蛋白的磷酸化，而且为神经退行性疾病中具有挑战性的病理蛋白修饰的特征描述提供了分析工作流程。
