Service de Pharmacologie et d'Immunoanalyse (SPI), Laboratoire d'Etude du Métabolisme des Médicaments (LEMM), CEA, INRA, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France.
Analyst. 2019 Oct 22;144(21):6342-6351. doi: 10.1039/c9an00751b.
Tau and α-synuclein are central in several neurodegenerative diseases, including Alzheimer Disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson Disease (PD). New analytical methods for precise quantification of cerebrospinal fluid (CSF) levels of both tau and α-synuclein are required to differentiate between dementias or monitor therapeutic responses. Notably, levels of total α-synuclein reported by ELISA are inconsistent among studies, impacted by antibody specificity or lack of standardization. Here, we report on the development and validation of a sensitive and robust mass spectrometry-based assay for the simultaneous quantification of tau and α-synuclein in CSF. The optimized workflow avoided any affinity reagents, and involved the combination of two enzymes, Glu-C and trypsin for optimal sequence coverage of α-synuclein acidic C-terminus. Up to 7 α-synuclein peptides were quantified, including the C-terminal peptide (132-140), resulting in a sequence coverage of 54% in CSF. The lower limits of quantification (LLOQ) ranged from 0.1 ng mL-1 to 1 ng mL-1 depending on the peptide. Regarding CSF tau, 4 peptides common to all isoforms were monitored, and LLOQ ranged from 0.5 ng mL-1 to 0.75 ng mL-1. The multiplex method was successfully applied to CSF samples from AD and DLB patients, two clinically overlapping neurodegenerative diseases. CSF α-synuclein levels were significantly lower in DLB patients compared to AD and controls. Moreover, tau and α-synuclein concentrations showed opposite trends in AD and DLB patients, suggesting the benefit of combining the two biomarkers for differentiation of DLB from AD and controls.
tau 和 α-突触核蛋白是几种神经退行性疾病的核心，包括阿尔茨海默病（AD）、路易体痴呆（DLB）和帕金森病（PD）。需要新的分析方法来精确量化脑脊液（CSF）中 tau 和 α-突触核蛋白的水平，以区分痴呆症或监测治疗反应。值得注意的是，ELISA 报告的总 α-突触核蛋白水平在研究之间不一致，受抗体特异性或缺乏标准化的影响。在这里，我们报告了一种灵敏且强大的基于质谱的方法的开发和验证，用于同时定量 CSF 中的 tau 和 α-突触核蛋白。优化的工作流程避免了任何亲和试剂，并涉及两种酶，Glu-C 和胰蛋白酶的组合，以实现 α-突触核蛋白酸性 C 端的最佳序列覆盖。多达 7 个 α-突触核蛋白肽被定量，包括 C 端肽（132-140），导致 CSF 中的序列覆盖率为 54%。取决于肽的不同，定量下限（LLOQ）范围为 0.1 ng mL-1 至 1 ng mL-1。关于 CSF tau，监测了所有同工型共有的 4 个肽，LLOQ 范围为 0.5 ng mL-1 至 0.75 ng mL-1。该多重方法成功应用于 AD 和 DLB 患者的 CSF 样本，这两种疾病在临床上有重叠。与 AD 和对照组相比，DLB 患者的 CSF α-突触核蛋白水平显著降低。此外，AD 和 DLB 患者的 tau 和 α-突触核蛋白浓度呈相反趋势，表明将这两种生物标志物结合起来用于区分 DLB 与 AD 和对照组具有益处。
