Background Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation of the tyrosine residue at position 39 (Y39) of α-synuclein (α-syn) (pY39 α-syn), which contributes to the death of dopaminergic neurons. Because pY39 α-syn may be pathogenic, monitoring pY39 α-syn could allow us to diagnose presymptomatic PD and help monitor disease progression and response to treatment. We sought to investigate if increased phosphorylation of pY39 α-syn can be detected in the cerebrospinal fluid (CSF) of PD patients by targeted mass spectrometry. Methods Here, we report a two-step enrichment method in which phosphotyrosine peptides were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium dioxide (TiO2) beads to detect EGVLpYVGSK sequence derived from tyrosine 39 region of α- and β-synuclein (αβ-syn). Accurate quantification was achieved by adding a synthetic heavy version of pY39 αβ-syn peptide before enzymatic digestion. Results Using the developed enrichment methods and optimized parallel reaction monitoring (PRM) assays, we detected pY39 αβ-syn peptide in human CSF and demonstrated that the ratio of pY39 αβ-syn to Y39 αβ-syn was significantly increased in the CSF of patients with PD. Conclusions We anticipate that this optimized two-step enrichment-based PRM detection method will help monitor c-Abl activation in PD patients and can also be used to quantify other phosphotyrosine peptides of low abundance in biological samples.

中文翻译：

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开发一种用于定量人脑脊液中酪氨酸 39 磷酸化 α-和 β-突触核蛋白的新方法。

背景帕金森病（PD）是第二大流行的神经退行性疾病。可以帮助监测 PD 进展或对疾病调节剂的反应的生物标志物对于做出适当的治疗决策是非常宝贵的。此外，可用于区分 PD 与具有 PD 样症状的其他相关疾病的生物标志物将有助于准确诊断和治疗。C-Abl 酪氨酸激酶在 PD 中被激活，导致 α-突触核蛋白 (α-syn) (pY39 α-syn) 的第 39 位 (Y39) 处的酪氨酸残基磷酸化增加，这有助于多巴胺能神经元的死亡。因为 pY39 α-syn 可能是致病性的，监测 pY39 α-syn 可以让我们诊断症状前的 PD 并帮助监测疾病进展和对治疗的反应。我们试图研究是否可以通过靶向质谱在 PD 患者的脑脊液 (CSF) 中检测到 pY39 α-syn 磷酸化的增加。方法 在这里，我们报告了一种两步富集方法，其中首先用抗磷酸酪氨酸抗体富集磷酸酪氨酸肽，然后用二氧化钛 (TiO2) 珠进行第二轮富集，以检测源自 α- 酪氨酸 39 区的 EGVLpYVGSK 序列。和β-突触核蛋白（αβ-syn）。通过在酶消化之前添加合成的重质 pY39 αβ-syn 肽来实现准确的定量。结果 使用开发的富集方法和优化的平行反应监测 (PRM) 测定，我们在人脑脊液中检测到 pY39 αβ-syn 肽，并证明 PD 患者脑脊液中 pY39 αβ-syn 与 Y39 αβ-syn 的比例显着增加。结论 我们预计，这种优化的基于两步富集的 PRM 检测方法将有助于监测 PD 患者的 c-Abl 活化，还可用于量化生物样品中其他低丰度的磷酸酪氨酸肽。