Proteomics

Hippocampi from 4 adults (P30; 2 males and 2 females) wild type (C57Bl/6J; JAX) mice were lysed in ice-cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% NP-40 and proteinase inhibitors) for 30 min. The supernatant was precleared by protein G agarose bead for 30 min prior to incubation with Sestd1 antibody overnight in cold room. Proteins associated with Sestd1 were then pulled down by protein G agarose bead for 3 h. Non-specific binders were removed by 3 washes in lysis buffer and 2 additional washes in 50 mM NH4HCO3, pH 7.5. Washed beads were then digested overnight with trypsin. The resulting peptides were desalted on Waters Sep-Pak C18 cartridges. Peptides were measured by nano-LC–MS-MS on a Thermo Scientific Q Exactive. Peptides were separated by reverse phase chromatography in a 180 min gradient (1–45% acetonitrile) at 250 nL/min. The Q Exactive was operated in the data-dependent mode with the following settings: 70 000 resolution, 300–2000 m/z full scan, Top 10, and an 1.8 m/z isolation window. Identification and label free quantification of peptides was done with MaxQuant 1.3.0.5 using a 1% false discovery rate (FDR) against the mouse Swiss-Prot/TrEMB database downloaded from Uniprot on 11 October 2013 (Cox and Mann 2008).