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Quantification of histone H1 subtypes using targeted proteomics

ABSTRACT: Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes: H1.0, H1.1-H15, and H1X. The variability in the proportions of somatic H1s (H1 complement) is one evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. These reasons led us to develop a mass spectrometry based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot, indicating the accuracy of the results. We used PRM to characterize the H1 complement in peripheral blood samples of healthy individuals, finding that the more abundant subtypes were H1.4 and H1.5. Analysis by PRM of samples from chronic myeloid leukemia patients showed that higher levels of H1 were associated with imatinibresistance, suggesting its potential as a predictive biomarker. Non-responder patients had lower proportions of H1.0, H1.1, and H1X than responders, hinting that their alteration could be involved in the acquisition of resistance.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood

DISEASE(S): Chronic Myeloid Leukemia

SUBMITTER: Marta Vilaseca

LAB HEAD: Marta Vilaseca

PROVIDER: PXD050243 | Pride | 2024-11-05

REPOSITORIES: Pride

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			Action	DRS
	3607_AR_GHstV7_Curve_10uL_R03.raw	Raw
	3607_AR_GHstV7_Curve_12uL_R03.raw	Raw
	3607_AR_GHstV7_Curve_15uL_R03.raw	Raw
	3607_AR_GHstV7_Curve_1uL_R03.raw	Raw
	3607_AR_GHstV7_Curve_2uL_R03.raw	Raw

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Project 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 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.

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Quantification of histone H1 subtypes using targeted proteomics

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