10.1021/pr2010814.s001 Nicholas T. Seyfried Nicholas T. Seyfried Yair M. Gozal Yair M. Gozal Laura E. Donovan Laura E. Donovan Jeremy H. Herskowitz Jeremy H. Herskowitz Eric B. Dammer Eric B. Dammer Qiangwei Xia Qiangwei Xia Li Ku Li Ku Jianjun Chang Jianjun Chang Duc M. Duong Duc M. Duong Howard D. Rees Howard D. Rees Deborah S. Cooper Deborah S. Cooper Jonathan D. Glass Jonathan D. Glass Marla Gearing Marla Gearing Malú G. Tansey Malú G. Tansey James J. Lah James J. Lah Yue Feng Yue Feng Allan I. Levey Allan I. Levey Junmin Peng Junmin Peng Quantitative Analysis of the Detergent-Insoluble Brain Proteome in Frontotemporal Lobar Degeneration Using SILAC Internal Standards American Chemical Society 2012 tandem mass spectrometry FTLD cases SILAC Internal StandardsA hallmark frontotemporal lobar degeneration FTLD brain tissue TDP PSF Frontotemporal Lobar Degeneration HEK SC RNA protein LC kidney 293 cells 2012-05-04 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Quantitative_Analysis_of_the_Detergent_Insoluble_Brain_Proteome_in_Frontotemporal_Lobar_Degeneration_Using_SILAC_Internal_Standards/2525788 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 21 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.