Quantitative proteomics of detergent insoluble tangles derived from Alzheimer's disease brains. (December 2021)

• Record Type: Journal Article
• Authors: Mukherjee, Soumya; Dubois, Celine; Perez, Keyla; Nisbet, Rebecca M; Li, Qiao‐Xin; Varghese, Shiji; Lago, Larissa Cristina; Klatt, Stephan; Birchall, Ian; Jin, Liang; McLean, Catriona; Streltsov, Victor; Barnham, Kevin J; Vella, Laura J; Masters, Colin L
• Is Part Of: Alzheimer's & dementia. Volume 17(2021)Supplement 3
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-12
• Subjects: Alzheimer's disease -- Periodicals; Alzheimer Disease -- Periodicals; Dementia -- Periodicals; Démence; Maladie d'Alzheimer; Périodique électronique (Descripteur de forme); Ressource Internet (Descripteur de forme); 616.83
• DOI: 10.1002/alz.055040
• Languages: English
• ISSNs: 1552-5260

Abstract

Abstract: Background: Ionic detergent N‐laurly sarcosine (Sarkosyl) has been the general choice of extraction and enrichment of detergent‐insoluble protein aggregates from different neurodegenerative proteinopathies from human post‐mortem brains. Sarkosyl‐insoluble pellets from Alzheimer's disease (AD) brains yield fibrils made up of paired helical filaments (PHFs) and straight helical filaments (SHF) of microtubule associated protein tau (MAPT), component of the neurofibrillary tangles in AD. Recent complementary cryo‐EM images and proteomic analysis have provided structural as well as biochemical detail of the core of these fibrillar elements. However, topographic detail of the PHFs and SHFs are still not complete as sarkosyl‐insoluble pellets are also known to contain amyloid‐beta (Aβ), another key hallmark of AD and warrants further assessment. Method: 1 g of fresh frozen post‐mortem frontal cortex tissues from non‐demented (n = 10) and AD brains (n =10) were homogenized in PBS‐sucrose buffer. 1 % Sarkosyl solution was then used to solubilize membrane associated proteins followed by 1 hr ultra‐centrifugation at 100, 000 g to separate detergent insoluble aggregated proteins. Complementary tryptic and Lys‐N digestion of the sarkosyl pellet was performed. We used quantitative proteomics using stable isotope labelled peptides and N 15 labelled proteins to estimate the amount of Aβ and MAPT, respectively, in the sarkosyl soluble and insoluble pellet fractions. Negative stain‐TEM were also performed for these final insoluble pellets. Result: Quantitative estimates of the molar ratio in the AD brains were on average equimolar amount of Aβ and 0N4R tau in the sarkosyl‐insoluble pellets. Total percentage of phosphorylated tau, pT181 (35 fmol/mg brain) and pT217 (8.1 fmol/mg brain) were estimated at 51 % and 34 %, respectively, in the detergent‐insoluble pellet. Proteomic analysis revealed enrichment of disease relevant post‐translational modifications (PTMs) of tau and Ab. Negative stain‐TEM indicates micro‐heterogeneity in tangles morphology obtained from each individual patient. Conclusion: Our results indicate equimolar Ab to MTBR rich 0N4R tau in the detergent insoluble pellets derived from frontal cortex of AD brain.