Protein extraction. All steps of the protein extraction were performed on ice or at 4°C. Isolated hippocampal tissue was homogenized in lysis buffer containing 20 mM triethylammonium bicarbonate (TEAB; Fluka), 5% sodium deoxycholate (SDC; Sigma-Aldrich), and one tablet of protease inhibitor (Roche). Homogenates were boiled for 3 min and subsequently sonicated three times for 10 s, each time paused for 30 s. Samples were centrifuged to pellet tissue debris, and the protein concentration in the supernatant was measured with a bicinchoninic acid protein assay (Pierce) using bovine serum albumin as standard.

Protein digestion. Dithiotreitol (DTT) was added to 200 μg of the global protein fraction up to a final concentration of 20 mM DTT in the solution. Samples were incubated for 20 min at 55°C and then centrifuged for 1 min at 10,000g (20°C). The supernatant was transferred to filter devices (10 kDa molecular weight cut off, VWR) and filled up with digestion buffer (DB) containing 20 mM TEAB and 0.5% SDC. Next, the samples were centrifuged for 10 min at 10,000g (20°C), the flow-through was discarded, and 100 μl of 40 mM indoacetamide in DB was added to the filter devices followed by a 20-min incubation at room temperature in darkness. Afterward, samples were washed twice with DB, and trypsin solved in DB was added to yield a protein-to-trypsin ratio of 100:1. Samples were incubated overnight at 37°C. Filter devices were centrifuged for 20 min at 10,000g (20°C), and the flow-through was collected. Residual peptides were removed by another centrifugation step with DB. The flow-throughs of one sample were combined, and, first, an equal volume of ethyl acetate and subsequently trifluoroacetic acid (final concentration 0.5%) were added. The solution was mixed and centrifuged for 2 min at 14,000g (20°C). The upper layer was discarded, and 250 μl of ethyl acetate was added, followed by centrifugation. The upper layer was discarded again, and the aqueous phase was collected for further procession.

Isobaric labeling and peptide fractionation. Peptides were vacuum concentrated and labeled with amine-reactive, six-plex tandem mass tag reagents (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer’s instructions. The labeling reaction was quenched by the addition of 5% hydroxylamine. Labeled peptides were pooled and desalted on Oasis HLB cartridges (Waters GmbH, Eschborn, Germany). Eluates containing 70% acetonitrile and 0.1% formic acid were dried and fractionated to 24 fractions by isoelectric point using an OFFGEL fractionator according to the manufacturer’s recommendations (Agilent Technologies, Waldbronn, Germany). Peptide fractions were dried and stored at −20°C.

Liquid chromatography–mass spectrometry analysis. Peptides were dissolved in 8 μl of 0.1% trifluoroacetic acid, and 1.5 μl was injected onto a C18 trap column (20 mm in length, 100 μm in inner diameter) coupled to a C18 analytical column (200 mm in length, 75 μm in inner diameter), made in house with 1.9 μm of ReproSil-Pur 120 C18-AQ particles (Dr. Maisch, Ammerbuch, Germany). Solvent A was 0.1% formic acid. Peptides were separated during a linear gradient from 4 to 40% solvent B (90% acetonitrile and 0.1% formic acid) within 90 min. The nano–high-performance liquid chromatography was coupled online to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptide ions between 330 and 1800 mass/charge ratio (m/z) were scanned in the Orbitrap detector with a resolution of 30,000 (maximum fill time of 400 ms, AGC (automatic gain control) target of 106, and lock mass of 371.0318 Da). The 20 most intense precursor ions (threshold intensity of 5000) were subjected to higher energy collision–induced dissociation, and fragments were also analyzed in the Orbitrap. Fragmented peptide ions were excluded from repeat analysis for 15 s. Raw data processing and analyses of database searches were performed with Proteome Discoverer software (Thermo Fisher Scientific). Peptide identification was done with an in-house Mascot server version 2.4.1 (Matrix Science Ltd., London, UK). MS2 data (including a-series ions) were searched against mouse sequences from Swiss-Prot (release 2014_01). Precursor ion m/z tolerance was 10 parts per million; fragment ion tolerance was 20 mmu (milli mass unit). Tryptic peptides were searched with up to two missed cleavages. Low scoring spectrum matches were searched again with semitryptic specificity with up to one missed cleavage. Oxidation (Met), acetylation (protein N terminus), and TMTsixplex (on Lys and N terminus) were set as dynamic modifications; carbamidomethylation (Cys) was set as static modification. Mascot results from searches against Swiss-Prot were sent to the percolator algorithm (70) version 2.04, as implemented in Proteome Discoverer. Only proteins with two peptides (maximum FDR of 1%) were considered as identified.

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