Quantitative Analysis
of the Detergent-Insoluble Brain Proteome in Frontotemporal Lobar
Degeneration Using SILAC Internal Standards
Nicholas T. Seyfried
Yair M. Gozal
Laura E. Donovan
Jeremy H. Herskowitz
Eric B. Dammer
Qiangwei Xia
Li Ku
Jianjun Chang
Duc M. Duong
Howard D. Rees
Deborah S. Cooper
Jonathan D. Glass
Marla Gearing
Malú
G. Tansey
James J. Lah
Yue Feng
Allan I. Levey
Junmin Peng
10.1021/pr2010814.s001
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.
2012-05-04 00:00:00
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