Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP,Brain Pathology

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Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP
Brain Pathology ( IF 5.8 ) Pub Date : 2020-12-10 , DOI: 10.1111/bpa.12923
Emily Feneberg ₁ , Philip D Charles ₂ , Mattéa J Finelli ₁ , Connor Scott ₁ , Benedikt M Kessler ₂ , Roman Fischer ₂ , Olaf Ansorge ₁ , Elizabeth Gray ₁ , Kevin Talbot ₁ , Martin R Turner ₁

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The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson’s, 8 Alzheimer’s disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6–100) and specificity of 100% (CI 68–100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70–100) and specificity of 100% (CI 68–100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

中文翻译：

ALS-TDP 中新型 C 末端 TDP-43 片段的检测和定量

肌萎缩侧索硬化症 (ALS) 的病理标志是存在细胞质包涵体，其中包含蛋白质 TDP-43 的 C 末端片段。在这里，我们检验了这样一个假设，即具有平行反应监测 (MS-PRM) 的高灵敏度质谱可以生成病理 TDP-43 肽比率的高分辨率图，从而形成定量 C 端 TDP-43 异常片段富集的基础。 . 对人体皮层和脊髓进行了生化分级分离，并通过免疫印迹和 MS 分析了 p-TDP-43、p-tau、α-突触核蛋白和 β-淀粉样蛋白病理学的存在，以检测全长和截断（疾病特异性）TDP-43 肽。这为通过 MS-PRM 对 16 名 ALS、8 名帕金森病、8 名帕金森病、8 例阿尔茨海默病和 8 例老年对照病例。我们通过免疫印迹证实了先前描述的 ALS-TDP 尿素组分中病理性 C 末端片段的富集。随后的 MS 分析解析了特定的 TDP-43 N 和 C 末端肽，包括一种新的 N 末端截短位点特异性肽。与正常和疾病对照相比，通过 MS-PRM 对肽进行的绝对定量显示，ALS-TDP 脑中的 C:N 末端 TDP-43 肽比率增加。AC:N 末端比率 >1.5 区分 ALS 与对照组的敏感性为 100% (CI 79.6-100) 和特异性为 100% (CI 68-100)，区分帕金森病和阿尔茨海默病的敏感性为 93% (CI 70–100) 和 100% (CI 68–100) 的特异性。ALS 中 N 端截短位点特异性肽的增加与 C 端片段富集一致，但在部分 C:N 末端比例正常但同时存在边缘 TDP-43 神经病理学变化的阿尔茨海默氏症病例中也发现了这种情况。总之，这是一种新的、灵敏的和特异性的方法来量化人脑中病理性 TDP-43 片段的富集，这可以构成无抗体检测的基础。我们的方法有可能帮助澄清特定的病理 TDP-43 肽特征是否与原发性或继发性 TDP-43 蛋白病相关。

更新日期：2020-12-10

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