title: Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards.
abstract: 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.
authors.affiliation: Department of Biochemistry, Center for Neurodegenerative Disease, School of Medicine, Emory University, Atlanta, Georgia 30322, USA. nseyfri@emory.edu
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.forename: Yair M
authors.initials: YM
authors.lastname: Gozal
authors.forename: Laura E
authors.initials: LE
authors.lastname: Donovan
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Herskowitz
authors.forename: Eric B
authors.initials: EB
authors.lastname: Dammer
authors.forename: Qiangwei
authors.initials: Q
authors.lastname: Xia
authors.forename: Li
authors.initials: L
authors.lastname: Ku
authors.forename: Jianjun
authors.initials: J
authors.lastname: Chang
authors.forename: Duc M
authors.initials: DM
authors.lastname: Duong
authors.forename: Howard D
authors.initials: HD
authors.lastname: Rees
authors.forename: Deborah S
authors.initials: DS
authors.lastname: Cooper
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Glass
authors.forename: Marla
authors.initials: M
authors.lastname: Gearing
authors.forename: Malú G
authors.initials: MG
authors.lastname: Tansey
authors.forename: James J
authors.initials: JJ
authors.lastname: Lah
authors.forename: Yue
authors.initials: Y
authors.lastname: Feng
authors.forename: Allan I
authors.initials: AI
authors.lastname: Levey
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D057174:Frontotemporal Lobar Degeneration / Q000378:metabolism / Q000473:pathology*; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism; D000072670:PTB-Associated Splicing Factor; D010766:Phosphorylation; D061251:Primary Cell Culture; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
chemical_list: D004268:DNA-Binding Proteins; D000072670:PTB-Associated Splicing Factor; D020543:Proteome; D016601:RNA-Binding Proteins