Towards complete ecdysteroidome of Drosophila melanogaster. Oksana Lavrynenko, Suzanne Eaton, Andrej Shevchenko. MPI CBG, Dresden, Germany.

   Ecdysone and 20-hydroxyecdysone control molting metamorphosis during Drosophila development ¹. Fruit flies are auxotrophs and produce ecdysteroids solely from dietary sterols. However, the exact relationship between hormone profiles and the sterol composition of food remains unclear ². Enzyme immunoassay (EIA) may estimate the total content of ecdysteroids, however it lacks specificity to distinguish and quantify individual hormones. We developed a LC-MS/MS strategy for ecdysteroidome (i.e. the full complement of ecdysteroid species) mapping. It relies on the targeted accurate mass screening, while species candidates are confirmed by MS/MS. Chemical modification of the total extract with Girard reagent followed by specific detection and fragmentation of candidate precursors enhanced the analysis specificity. In the series of proof-of -principle experiments we were able to identify polar conjugates (glycosides, phosphates and sulfates), catabolytes or any structurally modified hormones at the picograme level. We demonstrated that 20-hydroxyecdysone and makisterone A are synthesized independently from different precursors and could not be converted into each other. We also identified a new 20-hydroxyecdysone -related hormone that is highly enriched at the embryonic stage: previously, high ecdysteroid content was detected by EIA, but was not associated with any known hormone. Our approach provided further insight into the hormones catabolism pathways: we detected and quantified the carboxyl acids produced by C-26 specific oxidation of both 20-hydroxyecdysone and makisterone A acids. The new hormones and their metabolites could be quantified with acceptable accuracy with no recourse to synthetic standards. 1.Schwedes, C. C.; Carney, G. E., Ecdysone signaling in adult Drosophila melanogaster. J Insect Physiol 2012, 58 (3), 293-302. 2.Carvalho, M.; Schwudke, D.; Sampaio, J. L.; Palm, W.; Riezman, I.; Dey, G.; Gupta, G. D.; Mayor, S.; Riezman, H.; Shevchenko, A.; Kurzchalia, T. V.; Eaton, S., Survival strategies of a sterol auxotroph. Development 2010, 137 (21), 3675-3685.