Mouse hippocampus was homogenized in a buffer containing 2M NaCl, 10 mM HEPES/NaOH, 1 mM EDTA and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Homogenates were sonicated with an ultra-sonicator (Branson, Danbury, CT, USA) and centrifuged (16100 g, 20 min, 4 °C). The protein concentration was determined by Bradford assay.
Protein extracts were mixed with equal amounts of 15N-labeled DBA/2 mouse hippocampal protein extract24 . Fourty μg of 14N- 15N hippocampal protein mixture was separated in a 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250 (BioRad, Hercules, CA, USA) overnight. After destaining and cutting the gel lane into slices, tryptic peptides were produced and extracted as previously described42 (link). Extracted peptides were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) using a nanoflow HPLC-2D system (Eksigent, Dublin, California) coupled online to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Protein identification and quantitation were performed as described previously22 (link).
Protein extracts were mixed with equal amounts of 15N-labeled DBA/2 mouse hippocampal protein extract24 . Fourty μg of 14N- 15N hippocampal protein mixture was separated in a 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250 (BioRad, Hercules, CA, USA) overnight. After destaining and cutting the gel lane into slices, tryptic peptides were produced and extracted as previously described42 (link). Extracted peptides were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) using a nanoflow HPLC-2D system (Eksigent, Dublin, California) coupled online to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Protein identification and quantitation were performed as described previously22 (link).
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