Proteomics of Brain Proteome Revealed Altered Mitochondrial Function in Alzheimer Disease
A discovery-driven approach with isobaric tag for relative and absolute quantitation (iTRAQ) and label-free quantitative proteomics was used to profiled the mitochondrial proteome in human brain tissues of healthy and AD individuals.
Sample Processing Protocol
Frozen individual brain samples were thawed on ice and equal quantity was pooled in a group-wise manner. The individual clinical details of the patients are listed in Table 1. The brain tissue samples from each patient and control (in equal quantity) were pooled together within the group. We pooled two brain tissues in early onset AD and their age-matched control while old onset AD and control include five-tissue specimen. The tissue samples were homogenized. in a urea-acetate buffer (8M urea, 100 mM ammonium acetate, pH 6.0) to prevent asparagine deamidation. The cell debris was removed by centrifugation at 5000 x g for 7 min at 4º C. The proteins were purified by acetone precipitation and 200 µg proteins from each group were loaded into 12.5% SDS-PAGE and run at 80V for 45 min. Each sample lane was excised separately, cut into small pieces (approximately 1 mm2 pieces) and washed with 75% acetonitrile (ACN) containing triethylammonium bicarbonate (TEAB, 25 mM). The gel pieces were reduced with Tris 2- carboxyethyl phosphine hydrochloride (5 mM) and then alkylated with methyl methanethiosulfonate (10 mM). Gel pieces were dehydrated using ACN and subjected to protein digestion with sequencing grade modified trypsin (Promega, Madison, WI) at 37°C. The peptides were extracted using 50% acetonitrile, 5% acetic acid and concentrated using vacuum concentrators (Eppendorf AG, Hamburg, Germany).
Data Processing Protocol
Acquired data was further processed using Proteome Discoverer (PD) v1.4 (Thermo Scientific, San Jose, USA) with deisotope in MS/MS. The raw files were directly imported into the PD and further processed using designed workflow.Database search was performed against the UniProt human database.
Adav SS, Park JE, Sze SK. Quantitative profiling brain proteomes revealed mitochondrial dysfunction in Alzheimer's disease. Mol Brain. 2019 12(1):8 PubMed: 30691479