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We have undertaken a systematic proteomic approach to identify classical and non-classical secreted proteins which are produced in adipocyte cultures and adipose tissue explants. Serum-free primary culture was used, in association with tandem mass spectrometry, to characterize the secreted proteins during a time-course. In the 18 days of culture, a total of 281 proteins were found from adipocyte cultures or adipose tissue explants. Our method discovered 3 novel secreted proteins from adipocyte cultures: afamin (AFAM_RAT), seminal vesicle secretory protein 2 (SVS2_RAT) and xanthine dehydrogenase/oxidase (XDH_RAT). A further 9 novel secreted proteins were discovered from adipose tissue explants: chymase (CMA1_RAT), matrix Gla protein (MGP_RAT), biglycan (PGS1_RAT), serine protease inhibitor A3K (SPA3K_RAT), serine protease inhibitor A3L (SPA3L_RAT), serine protease inhibitor A3M (SPA3M_RAT), serine protease inhibitor A3N (SPA3N_RAT), SPARC-like protein 1 (SPRL1_RAT) and seminal vesicle secretory protein 2 (SVS2_RAT). Previous studies suggested that these proteins may play a role in tissue development in association with obesity or obesity-related conditions, giving them potential as biomarker of adipocyte-associated disease. In this regard, we show how our data could be used to create a reliable SRM/MRM database that would be applicable to early diagnosis of adipocyte-associated disease.