Computational protocol: The adipocyte differentiation protein APMAP is an endogenous suppressor of Aβ production in the brain

Similar protocols

Protocol publication

[…] The γ-secretase complex was purified as previously described () and was run on a NativePAGE Novex® Bis–Tris 4–16% gel for BN-PAGE analysis (Invitrogen, Carlsbad, CA, USA). Next, the complex was visualized by Coomassie staining, excised and cut into small pieces. Proteins were reduced, alkylated and subjected to in-gel digestion with trypsin. Briefly, gel pieces were destained, desiccated by incubating twice in 200 µl of 50 mm ammonium bicarbonate and 50% ethanol for 20 min and dried with a vacuum concentrator. The samples were then incubated overnight at 37°C with trypsin (12.5 ng/µl). For liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis after extraction from gel slices, peptides were resuspended in 2% Acetonitril/0.1% Formic Acid and separated by reverse-phase chromatography on a Dionex Ultimate 3000 RSLC nanoUPLC system connected in-line with an Orbitrap Elite (Thermo Fischer Scientific, Waltham, MA, USA). The instrument was operated in an information-dependent mode where peptide masses were selected for collision-induced dissociation to generate tandem mass spectra. A database search was performed using Mascot 2.3 (Matrix Science, Boston, MA, USA) and SEQUEST in Proteome Discoverer v.1.3 against a human database (UniProt release 2013_01; 87613 sequences) and Mammalian database (UniProt release 2011_07; 91104 sequences). All searches were performed with trypsin cleavage specificity, with up to three missed cleavages allowed, an ion mass tolerance of 10 ppm for the precursor and 0.5 Da for the fragments. Carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (protein N-term) and phosphorylation (STY) were considered variable modifications. Data were further processed and inspected using Scaffold 3 software (Proteome Software). […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Alzheimer Disease, Sandhoff Disease