We investigated the age-associated thermogenesis activation in brown fat mitochondria. We administered the young mice (10 weeks old) and old mice (76 weeks old) with a selective β3-adrenergic receptor agonist (β3-AR agonist, CL316,243) at a high dose (1 mg kg-1) for five days at 30˚C to activate thermogenesis. We isolated mitochondria from brown fat for proteome profiling. We applied 250 ng peptides for LC/MS/MS analysis using label-free quantification coupled match-between-run approach.