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. 2009 Nov;8(11):5069-79.
doi: 10.1021/pr900474t.

Proteomics analysis reveals novel components in the detergent-insoluble subproteome in Alzheimer's disease

Yair M Gozal  1 , Duc M Duong, Marla Gearing, Dongmei Cheng, John J Hanfelt, Christopher Funderburk, Junmin Peng, James J Lah, Allan I Levey
Affiliations

Proteomics analysis reveals novel components in the detergent-insoluble subproteome in Alzheimer's disease

Yair M Gozal et al. J Proteome Res. 2009 Nov.

Abstract

Neurodegenerative diseases are often defined pathologically by the presence of protein aggregates. These aggregates, including amyloid plaques in Alzheimer's disease (AD), result from the abnormal accumulation and processing of proteins, and may ultimately lead to neuronal dysfunction and cell death. To date, conventional biochemical studies have revealed abundant core components in protein aggregates. However, rapidly improving proteomics technologies offer opportunities to revisit pathologic aggregate composition, and to identify less abundant but potentially important functional molecules that participate in neurodegeneration. The purpose of this study was to establish a proteomic strategy for the profiling of neurodegenerative disease tissues for disease-specific changes in protein abundance. Using high resolution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), we analyzed detergent-insoluble frontal cortex samples from AD and unaffected control cases. In addition, we analyzed samples from frontotemporal lobar degeneration (FTLD) cases to identify AD-specific changes not present in other neurodegenerative diseases. We used a labeling-free quantification technique to compare the abundance of identified peptides in the samples based on extracted ion current (XIC) of their corresponding ions. Of the 512 identified proteins, quantitation demonstrated significant changes in 81 AD-specific proteins. Following additional manual filtering, 11 proteins were accepted with high confidence as increased in AD compared to control and FTLD brains, including beta-amyloid, tau and apolipoprotein E, all well-established AD-linked proteins. In addition, we identified and validated the presence of serine protease 15, ankyrin B, and 14-3-3 eta in the detergent-insoluble fraction. Our results provide further evidence for the capacity of proteomics applications to identify conserved sets of disease-specific proteins in AD, to enhance our understanding of disease pathogenesis, and to deliver new candidates for the development of effective therapies for this, and other, devastating neurodegenerative disorders.

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Figures

Fig. 1
Fig. 1
Diagram of sequential extraction protocol used to generate detergent-insoluble fractions for proteomic analysis.
Fig. 2
Fig. 2. Sample preparation for proteomic analysis
(A) Diagram of strategy for sample preparation and “bottom-up” proteomics analysis in which proteins are digested into peptides for improved separation and ionization. (B) SDS-PAGE gel of the isolated urea fractions stained with Coomassie Blue G-250. The gel lanes were excised in 5 pieces as indicated.
Fig. 3
Fig. 3. Statistical evaluation and filtering of proteomics data
(A) Abundance ratios for AD/Control comparison for all proteins across all gel bands were transformed (logarithmic base 2) and plotted with each point corresponding to the number of proteins in 0.3 unit windows (black line). A Gaussian curve was subsequently fitted to the data (red line) and used to determine significance levels for protein change. (B) Fitted normal distributions for all possible case comparisons with (C) statistical means, standard deviations, and regression coefficients. (D) Additional filtering criteria for removal of false positives were based on quality assessment of protein quantification by manual scoring of corresponding peptides. A top scoring protein would receive 1 point in each of the above categories for a total of 3 points. SN, Signal to Noise Ratio.
Fig. 3
Fig. 3. Statistical evaluation and filtering of proteomics data
(A) Abundance ratios for AD/Control comparison for all proteins across all gel bands were transformed (logarithmic base 2) and plotted with each point corresponding to the number of proteins in 0.3 unit windows (black line). A Gaussian curve was subsequently fitted to the data (red line) and used to determine significance levels for protein change. (B) Fitted normal distributions for all possible case comparisons with (C) statistical means, standard deviations, and regression coefficients. (D) Additional filtering criteria for removal of false positives were based on quality assessment of protein quantification by manual scoring of corresponding peptides. A top scoring protein would receive 1 point in each of the above categories for a total of 3 points. SN, Signal to Noise Ratio.
Fig. 4
Fig. 4. Confirmation of proteomic candidates by immunoblot analysis
Frontal cortex from FTLD-U, AD, and unaffected control cases were pooled by diagnosis and sequentially extracted with buffers containing triton X-100, sarkosyl, and urea. Sarkosyl insoluble (urea) samples were immunoblotted with antibodies to (A) Tau2 which recognizes both non-phosphorylated and abnormal phosphorylated tau (region shown ~30kD–60kD) (B) apolipoprotein E (36kD denoted by arrow) (C) 14-3-3η (28kD denoted by arrow) (D) Serine protease 15 (106kD denoted by arrow) and (E) Ankyrin B (multiple isoforms denoted by arrows at 205kD, 163kD, and 100kD) (F) Representative Ponceau S reversible membrane stain was performed to ensure both equal loading and complete transfer of proteins from the gel. (G) Unpooled AD (n = 10) and control (n = 10) cases were individually detergent-extracted and immunoblotted with an antibody to 14-3-3η. The corresponding Ponceau S reversible membrane stain is shown. (H) Quantification of immunoreactive 14-3-3η bands revealed a 2.1-fold enrichment of this protein in AD urea extract (p < 0.01).
Fig. 5
Fig. 5. Confirmation of proteomic candidates by immunohistochemistry in frontal cortex floating sections (50μm)
(A) Apolipoprotein E staining in control tissue (200μm) and (B) AD (100μm). (B) Ankyrin B staining in control tissue (200μm) and (B) AD (200μm). (C) 14-3-3η staining in control tissue (200μm) and (B) AD (100μm). (Scale Bar in μm)
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