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Abstract
Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.
Highlights
Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods
Methods have been developed for detecting individual endogenous insect ecdysteroids by GC-MS [19] and Abbreviations: CID, collision-induced dissociation; E, ecdysone; G(c), Girard C reagent; G(p), Girard P reagent; G(t), Girard T reagent; G(x)Y, hydrazone of the ecdysteroid Y and Girard reagent G(x) [in this notation, G(p)E is the hydrazone of ecdysone and Girard P reagent]; dh-G(p)E, water loss product of G(p)E; MRM, multiple reaction monitoring; Py, pyridine; TMA, trimethylamine; 20H, 20-hydroxyecdysone
Preliminary experiments performed under conventional reaction conditions [25] showed that the 6-ketone group of ecdysone was much less reactive toward hydrazides compared with 3-ketosterols
Summary
Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. Ecdysone (E), 20-hydroxyecdysone (20H), and makisterone A (Fig. 1 and supplementary Fig. I) are major ecdysteroids in Drosophila Their content in individual animals depends on both the developmental stage and the availability of dietary sterols [8, 9]. Apart from these three major hormones, several low abundant molecules, including 20-deoxy-makisterone A [9], 24-epi-20-deoxy-makisterone A [8], and 20-hydroxyecdysone-22-acetate [10] have been characterized; the existence of other ecdysteroid-related structures has been inferred from indirect biological evidence [11]. Unequivocal identification of known and discovery of novel endogenous ecdysteroids require alternative approaches relying on molecular forms having more specific fragmentation pathways
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