Fig. 1.

Metabolic labeling in mice and identification of synaptic LLPs. (A, Left) Schematic of metabolic labeling in mice. At 3 wk old, male mice were fed with chow containing heavy-isotope-labeled Lys-¹³C₆ for 7 wk, followed by unlabeled chow for 7 wk. Mice were exposed to either control or EE for 3 d before chasing with unlabeled chow. Mice were killed at the end of the pulse or chase periods followed by tissue isolation, subcellular fractionation, and analysis by LC-MS/MS. (A, Right) Stable proteins incorporate less label during pulse but retain more during chase. Unstable proteins rapidly acquire and then lose heavy label. (B) RIA of heavy lysine incorporated in the proteome during the pulse of forebrain synaptosomes (Syn.), cytosol, and or whole kidney. Box plot shows high levels of heavy labeling were achieved on average; individual protein species show variable labeling according to their turnover. Labeling efficiency was significantly higher in the kidney compared with brain, indicating higher rates of protein metabolism (P < 0.05, Student’s t test). Data obtained from eight mice. Error bars indicate the full distribution of data points. (C) Subcellular fractionation of mouse forebrain was monitored by Western blot analyses of synaptic and nonsynaptic proteins. Whole forebrain lysate, cytosol, and synaptosomal fractions (Syn.) were analyzed.