Journal
BRAIN PATHOLOGY
Volume 31, Issue 4, Pages -Publisher
WILEY
DOI: 10.1111/bpa.12923
Keywords
Alzheimer's disease; amyotrophic lateral sclerosis; LATE; proteomic biomarker; TDP-43
Categories
Funding
- Medical Research Council & Lady Edith Wolfson MND Association Clinical Training Fellowship [MR/R000743/1]
- Guarantors of Brain Clinical Post Doctorial Fellowship
- Alzheimer's Research UK [AVR01961]
- Motor Neurone Disease Association
- John Fell Fund [133/075]
- Wellcome [097813/Z/11/Z]
- Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China [2018-I2M-2-002]
- National Institute for Health Research UK
- Medical Research Council UK
- Brains for Dementia Research UK
- Wellcome Trust [097813/Z/11/Z] Funding Source: Wellcome Trust
- MRC [MR/K01014X/1, MR/R000743/1, G0701923] Funding Source: UKRI
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The study used mass spectrometry with parallel reaction monitoring (MS-PRM) to quantify pathological TDP-43 fragments in the brain of ALS patients. The increased C:N-terminal TDP-43 peptide ratio in ALS patients can distinguish them from controls and other diseases. N-terminal truncation site-specific peptides were elevated in ALS patients and some Alzheimer cases with limbic TDP-43 neuropathological changes.
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.
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