Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila

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. 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. Ecdysteroids are a large family of structurally diverse polyhydroxylated sterols found in plants and animals of the Arthropoda phylum (reviewed in Ref. 1Gilbert L.I. Insect Endocrinology. 1st edition. Elsevier/Academic Press, London; Waltham, MA2012Google Scholar). In insects, ecdysteroids control life-cycle progression, including molting, reproduction, stress response, and lifespan, via conserved regulatory pathways (reviewed in Ref. 2Schwedes C.C. Carney G.E. Ecdysone signaling in adult Drosophila melanogaster.J. Insect Physiol. 2012; 58: 293-302Crossref PubMed Scopus (108) Google Scholar). The fruit fly Drosophila melanogaster is an established model organism in developmental biology and now is becoming an important system for studying lipid metabolism and its hormonal regulation (3Baker K.D. Thummel C.S. Diabetic larvae and obese flies-emerging studies of metabolism in Drosophila.Cell Metab. 2007; 6: 257-266Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 4Brogiolo W. Stocker H. Ikeya T. Rintelen F. Fernandez R. Hafen E. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control.Curr. Biol. 2001; 11: 213-221Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 5Spindler K.D. Honl C. Tremmel C. Braun S. Ruff H. Spindler-Barth M. Ecdysteroid hormone action.Cell. Mol. Life Sci. 2009; 66: 3837-3850Crossref PubMed Scopus (91) Google Scholar–6Gutierrez E. Wiggins D. Fielding B. Gould A.P. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism.Nature. 2007; 445: 275-280Crossref PubMed Scopus (297) Google Scholar). Drosophila is a sterol auxotroph, and its ste