Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP
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-res...
| Main Authors: | , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Wiley
2021
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| _version_ | 1826288101472862208 |
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| author | Feneberg, E Charles, P Finelli, MJ Scott, C Kessler, B Fischer, R Ansorge, O Gray, E Talbot, K Turner, M |
| author_facet | Feneberg, E Charles, P Finelli, MJ Scott, C Kessler, B Fischer, R Ansorge, O Gray, E Talbot, K Turner, M |
| author_sort | Feneberg, E |
| collection | OXFORD |
| description | 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 phosphoTDP-43, p-tau, alpha-synuclein and beta-amyloid pathology, were biochemically fractionated and analysed by immunoblot and MS for detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed synthesis of heavy isotope-labelled peptides for the absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson’s and 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 TDP-43 pathology.
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. |
| first_indexed | 2024-03-07T02:08:38Z |
| format | Journal article |
| id | oxford-uuid:9fd7c668-61d0-4047-b9b2-b941062df841 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T02:08:38Z |
| publishDate | 2021 |
| publisher | Wiley |
| record_format | dspace |
| spelling | oxford-uuid:9fd7c668-61d0-4047-b9b2-b941062df8412022-03-27T02:01:05ZDetection and quantification of novel C-terminal TDP-43 fragments in ALS-TDPJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:9fd7c668-61d0-4047-b9b2-b941062df841EnglishSymplectic ElementsWiley2021Feneberg, ECharles, PFinelli, MJScott, CKessler, BFischer, RAnsorge, OGray, ETalbot, KTurner, MThe 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 phosphoTDP-43, p-tau, alpha-synuclein and beta-amyloid pathology, were biochemically fractionated and analysed by immunoblot and MS for detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed synthesis of heavy isotope-labelled peptides for the absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson’s and 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 TDP-43 pathology. 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. |
| spellingShingle | Feneberg, E Charles, P Finelli, MJ Scott, C Kessler, B Fischer, R Ansorge, O Gray, E Talbot, K Turner, M Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP |
| title | Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP |
| title_full | Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP |
| title_fullStr | Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP |
| title_full_unstemmed | Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP |
| title_short | Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP |
| title_sort | detection and quantification of novel c terminal tdp 43 fragments in als tdp |
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