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. 2019 Jul 19:14 Pt B:66-73.
doi: 10.1016/j.clinms.2019.07.003. eCollection 2019 Nov.

Detection and characterization of TDP-43 in human cells and tissues by multiple reaction monitoring mass spectrometry

Taylor D Pobran  1 , Lauren M Forgrave  1 , Yu Zi Zheng  1 , John G K Lim  1 , Ian R A Mackenzie  1   2 , Mari L DeMarco  1   3
Affiliations

Detection and characterization of TDP-43 in human cells and tissues by multiple reaction monitoring mass spectrometry

Taylor D Pobran et al. Clin Mass Spectrom. .

Abstract

Transactive response DNA-binding protein 43 kDa (TDP-43) is a highly conserved and widely expressed protein in human tissues that regulates nucleic acid processing. In frontotemporal dementia and amyotrophic lateral sclerosis, however, TDP-43 forms insoluble aggregates in central nervous tissues. These pathological deposits of TDP-43 have been primarily studied by ligand binding, namely western blot analysis, and, thus, methods with greater structural resolution are needed to aid in our understanding of the pathological processes associated with TDP-43 misfolding and aggregation. Toward this goal, we have developed a selective and multiplex method for the detection and characterization of TDP-43 using liquid chromatography tandem mass spectrometry. As proof-of-concept, the method was applied to the detection and characterization of TDP-43 in human cell lines and human brain tissue.

Keywords: ALS, amyotrophic lateral sclerosis; Amyotrophic lateral sclerosis; Biomarkers; FTD, frontotemporal dementia; FTLD, frontotemporal lobar degeneration; Frontotemporal dementia; LC, liquid chromatography; MRM, multiple reaction monitoring; MS/MS, triple quadrupole mass spectrometer; Mass spectrometry; Multiple reaction monitoring; PTM, post-translational modification; S/N, signal to noise ration; TDP-43, transactive response DNA-binding protein 43 kDa; Transactive response DNA-binding protein 43 kDa; ePAR, expected peak area ratio; p, detergent-insoluble fraction; rec, recombinant; s, detergent-soluble fraction.

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Conflict of interest statement

M.L.D. reports a grant from the Alzheimer's Drug Discovery Foundation and the Association for Frontotemporal Degeneration, and Y.Z.Z. reports a fellowship from the Alzheimer Society of Canada and the Firefly Foundation, during the conduct of the study. I.R.A.M. reports personal fees from Prevail Therapeutics, outside of the submitted work. T.D.P., L.M.F, and J.G.K.L. have nothing to disclose.

Figures

Fig. 1
Fig. 1
TDP-43 protein sequence and domains, with the location of proteotypic peptides included in the MRM LC–MS/MS method noted. (NLS: nuclear localization sequence; RRM: RNA recognition motif; NES: nuclear export sequence; GRR: glycine-rich region; Q/N: glutamine- and asparagine-rich region).
Fig. 2
Fig. 2
Representative chromatogram of the TDP-43 6-plex MRM assay.
Fig. 3
Fig. 3
Product ion scans of recTDP-43 tryptic peptides pre-optimization.
Fig. 4
Fig. 4
Linearity of the response of TDP252-263.
Fig. 5
Fig. 5
(A) SDS-PAGE and (B) TDP-43 western blot analysis of HeLa cell lysate and recTDP-43. Boxes indicate gel excisions subjected to in-gel digest and LC–MS/MS analysis.
Fig. 6
Fig. 6
(A) SDS-PAGE and (B) western blot analysis of unaffected and FTLD-TDP type A human brain tissue (dashed line: gel region excised for in-gel digestion and LC–MS/MS analysis).
See this image and copyright information in PMC

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