Quantitative proteomic analysis of the frontal cortex in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by intracellular formation of neurofibrillary tangles and extracellular deposition of β-amyloid protein (Aβ) in the extracellular matrix. The pathogenesis of AD has not yet been fully elucidated and little is known about global alterations in the brain proteome that are related to AD. To identify and quantify such AD-related changes in the brain, we employed a tandem mass tags approach coupled to high-resolution mass spectrometry. We compared the proteomes of frontal cortex from AD patients with corresponding age-matched brain samples. Liquid chromatography-mass spectrometry/MS analysis carried out on an Orbitrap Fusion Lumos Tribrid mass spectrometer led to identification of 8,066 proteins. Of these, 432 proteins were observed to be significantly altered (>1.5 fold) in their expression in AD brains. Proteins whose abundance was previously known to be altered in AD were identified including secreted phosphoprotein 1 (SPP1), somatostatin (SST), SPARC-related modular calcium binding 1 (SMOC1), dual specificity phosphatase 26 (DUSP26), and neuronal pentraxin 2 (NPTX2). In addition, we identified several novel candidates whose association with AD has not been previously described. Of the novel molecules, we validated chromogranin A (CHGA), inner membrane mitochondrial protein (IMMT) and RAS like proto-oncogene A (RALA) in an additional set of 20 independent brain samples using targeted parallel reaction monitoring mass spectrometry assays. The differentially expressed proteins discovered in our study, once validated in larger cohorts, should help discern the pathogenesis of AD.

Keywords: AD; TMT; neurodegeneration; proteome.

Figure 1.

A schematic of the workflow used to study the proteomic changes in the brain of AD patients. The brain samples were homogenized in liquid nitrogen and lysed in 2% SDS buffer. Brain proteomes from cognitively normal individuals were compared with that of AD patients using a TMT-based quantitative proteomics approach in the discovery step. Validation was carried out using parallel reaction monitoring (PRM) assays for a subset of molecules that were found to be altered in abundance in AD.

Figure 2.

Summary of TMT-based discovery experiments (A) PCA plot for the brain proteome data (B) Volcano plot for the proteome data for identification significantly altered proteins in AD brain (C) Heat map of the significantly altered proteins in AD brain (D) Distribution of fold-change values (log₂ ratios) (AD/Controls) for proteins identified in the study (E) Enriched altered signaling pathways in AD brain

Figure 3.

Representative MS/MS spectra of peptides identified along with quantitation provided in the box plot: The length of the box is thus the interquartile range of the sample. The other dimension of the box does not represent anything in particular. A line is drawn across the box at the sample median. Whiskers sprout from the two ends of the box until they reach the sample maximum and minimum. The crossbar at the far end of each whisker is optional and its length signifies nothing. A) Osteopontin (SPP1) along with its reported ion quantification shown in box plot with significant change with P value P < 0.0001 (B) Somatostatin (SST) along with its reported ion quantification shown in box plot with significant change with P value P < 0.0013 (C) Chromogranin A (CHGA) along with its reported ion quantification shown in box plot with significant change with P value P < 0.0045; and, D) Inner Membrane Mitochondrial Protein (IMMT) along with its reported ion quantification shown in box plot with significant change with P value P < 0.0009.

Figure 4.

PRM-based validation of candidate proteins by parallel reaction monitoring. The length of the box is thus the interquartile range of the sample. The other dimension of the box does not represent anything in particular. A line is drawn across the box at the sample median. Whiskers sprout from the two ends of the box until they reach the sample maximum and minimum. The crossbar at the far end of each whisker is optional and its length signifies nothing.: (A) Chromogranin A (CHGA); quantification shown in box plot with significant change with P value P < 0.0001 and ROC curve (B) Inner Membrane Mitochondrial Protein (IMMT); quantification shown in box plot with significant change with P value P < 0.0075 and ROC curve (C) Ras-related protein Ral-A (RalA); quantification shown in box plot with significant change with P value P < 0.04 and ROC curve