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Author Notes:

Corresponding Authors: Nicholas T. Seyfried, Tel. 404.712.8589, nseyfri@emory.edu and Junmin Peng Tel. 404.712.8510, jpeng@genetics.emory.edu

NTS and YMG contributed equally to this work

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Research Funding:

This work was supported by the National Institutes of Health through the Emory Alzheimer’s Disease Center grant (P50AG025688), the Emory Neuroscience NINDS Core Facilities (P30NS055077), and NIH training grants (F32AG032848–02 to N.T.S., 5T32NS007480 to E.B.D. and F30NS057902 to Y.M.G.).

Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards

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Journal Title:

Journal of Proteome Research

Volume:

Volume 11, Number 5

Publisher:

, Pages 2721-2738

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Summary A hallmark of neurodegeneration is the aggregation of disease related proteins that are resistant to detergent extraction. In the major pathological subtype of frontotemporal lobar degeneration (FTLD), modified TAR-DNA binding protein 43 (TDP-43), including phosphorylated, ubiquitinated and proteolytically cleaved forms, is enriched in detergent-insoluble fractions from post-mortem brain tissue. Additional proteins that accumulate in the detergent-insoluble FTLD brain proteome remain largely unknown. In this study, we used proteins from stable isotope-labeled (SILAC) human embryonic kidney 293 cells (HEK293) as internal standards for peptide quantitation across control and FTLD insoluble brain proteomes. Proteins were identified and quantified by liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS) and twenty-one proteins were determined to be enriched in FTLD using SILAC internal standards. In parallel, label free quantification of only the unlabeled brain derived peptides by spectral counts (SC) and G-test analysis identified additional brain-specific proteins significantly enriched in disease. Several proteins determined to be enriched in FTLD using SILAC internal standards were not considered significant by G-test due to their low total number of SC. However immunoblotting of FTLD and control samples confirmed enrichment of these proteins, highlighting the utility of SILAC internal standard to quantify low abundance proteins in brain. Of these, the RNA binding protein PTB-associated splicing factor (PSF) was further characterized because of structural and functional similarities to TDP-43. Full-length PSF and shorter molecular weight fragments, likely resulting from proteolytic cleavage, were enriched in FTLD cases. Immunohistochemical analysis of PSF revealed predominately nuclear localization in control and FTLD brain tissue and was not associated with phosphorylated pathologic TDP-43 neuronal inclusions. However, in a subset of FTLD cases, PSF was aberrantly localized to the cytoplasm of oligodendrocytes. These data raise the possibility that PSF directed RNA processes in oligodendrocytes are altered in neurodegenerative disease.

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© 2012 American Chemical Society

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