LGC Group, Queens Road, TW11 0LY Teddington, UK.
LBPC-PPC, Univ Montpellier, IRMB CHU Montpellier, INM INSERM, 34295 Montpellier, France.
Analyst. 2024 Sep 23;149(19):4842-4850. doi: 10.1039/d4an00634h.
α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.
α-突触核蛋白聚集是神经退行性疾病的重要标志，如帕金森病（PD）和路易体痴呆。α-突触核蛋白已越来越多地被用作 PD 和其他突触核蛋白病的诊断生物标志物。目前的临床检测方法依赖于基于抗体的免疫测定来检测α-突触核蛋白，这种方法具有高灵敏度、高通量和小样本量的特点。然而，这些检测方法的实用性可能会受到所使用的抗体的特异性、选择性和批次间异质性的影响，这可能导致在比较不同实验室的结果时，蛋白质总量的测量结果存在偏差。同样，目前基于质谱的α-突触核蛋白定量方法缺乏经过良好定义、赋值的校准品，以确保测量结果的可比性。因此，仍然需要通过开发利用与 SI（国际单位制）可溯源的校准品的参考测量程序（RMP）来实现对α-突触核蛋白的临床测量的标准化。在这里，我们报告了一种使用 SI 可溯源的初级校准品和同位素稀释质谱（IDMS）方法的候选 RMP，以满足这一需求。使用组合的氨基酸分析（AAA）和定量核磁共振（qNMR）进行可溯源定量，制备了重量法制备的初级校准品，并进行赋值。设计了一种优化的靶向样品净化程序，涉及非变性 Lys-C 消化和固相萃取策略，然后开发了一种用于定量脑脊液（CSF）中α-突触核蛋白的靶向多重反应监测（MRM）方法。然后，该候选 RMP 用于敏感检测和准确量化患者来源 CSF 样本中的多种蛋白型α-突触核蛋白肽。随后将基于 LC-MS 的结果与免疫测定数据进行比较，以评估我们方法的整体性能。该候选 RMP 的开发和采用，以及 SI 可溯源的初级校准品的可用性，将允许基于 LC-MS 的测定对 CSF 中的α-突触核蛋白进行可靠的定量。该 RMP 将有可能有助于该重要生物标志物的标准化，并可能导致未来的实验室间比较。
