Animals were anesthetized at indicated ages using phenobarbital and perfused transcardially with PBS. Brains were removed, with cortex and hippocampus subdissected and flash-frozen using liquid nitrogen. Cortex and hippocampus were combined and homogenized in homogenization buffer, followed by brief sonication. Tissue homogenate was centrifuged at 1500g for 5 min to remove cell debris, and protein concentration was measured with BCA reagent using BSA as standard. Experimental tissue homogenate (1000 μg) was mixed with 500 μg of brain homogenate from mice fed with feed containing l-lysine (13C6) (MT-LYSC6-MB-PK, Cambridge Isotope Laboratories Inc.) for spike-in SILAM analysis. SDD-AGE sample buffer was added to the mixed sample and incubated at room temperature for 10 min before loading onto a 1.6% low melting point agarose gel (01146, Nacalai Tesque Inc.) containing 1% SDS in tris-acetate-EDTA (TAE) buffer. After electrophoresis in TAE buffer containing 0.1% SDS, the gel piece above 250 kDa was excised, melted, and digested by thermostable β-agarase (31107121, Nippon Gene Co.). Proteins in this “high–molecular weight” fraction were prepared for enzymatic digestion by using the filter-aided sample preparation, as described previously (47). The protein sample was digested overnight at 37°C using Lys-C, with digested peptides eluted from the filter by centrifugation. For MS, 2 μg of peptides was analyzed by liquid chromatography–electrospray ionization–MS/MS (LC-ESI-MS/MS).

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