Quantitative proteomics of tau and Aβ in detergent fractions from Alzheimer's disease brains

Abstract

The two hallmarks of Alzheimer's disease (AD) are amyloidβ (Aβ) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aβ drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aβ mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aβ species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aβ 42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT 181 (~22%) and pT 217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold

Protein Interactome of Amyloid-β as a Therapeutic Target

“…Notably, tau phosphorylation is an ordinary event in cell physiology [ 53 , 54 ], and eight possible phosphorylation sites have been discovered on all six isoforms of tau [ 55 , 56 ]. The mechanisms of Aβ’s effect on tau hyperphosphorylation are not yet clear, despite the fact that there is much evidence of the two polypeptides’ coaggregation in the brains of AD patients [ 57 , 58 ]. In a recent report, the effect of Aβ on the kinases phosphorylating tau was evidenced [ 59 ], while earlier data suggest the possibility of targeting by Aβ available tau phosphorylation sites [ 60 ] ( Figure 2 A).…”

Establishing Quality Control Metrics for Large-Scale Plasma Proteomic Sample Preparation

“…11,12 LC−MS/MS methodologies can provide enhanced specificity, cost-effectiveness, and the ability to manage sample complexity and quantify thousands of proteins in a single MS run. 13 Biological samples, such as brain tissue, 14,15 single-cells, 16,17 or blood plasma, 18,19 are of particular interest, especially in the context of cardiovascular and neurodegenerative diseases. Plasma, which is often collected during routine clinical visits, remains a useful proxy of disease.…”

“…Quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS) approaches, in addition to immunoaffinity proximity extension assays (Olink) and aptamer-based assays (SomaScan), − allow researchers to measure relative protein levels in patient samples. − LC–MS/MS-based proteomics is a complementary antibody-based assay when profiling a patient’s clinical disease status. , LC–MS/MS methodologies can provide enhanced specificity, cost-effectiveness, and the ability to manage sample complexity and quantify thousands of proteins in a single MS run . Biological samples, such as brain tissue, , single-cells, , or blood plasma, , are of particular interest, especially in the context of cardiovascular and neurodegenerative diseases. Plasma, which is often collected during routine clinical visits, remains a useful proxy of disease …”