Drosophila Metabolite Quantification by LC-MS

LC-MS was performed as previous reported (Han et al., 2017 (link)). The Dionex Ultimate 3000 UPLC system was coupled to a TSQ Quantiva Ultra triple-quadrupole mass spectrometer (Thermo Fisher, Waltham, MA), equipped with a heated electrospray ionization probe in negative ion mode. Extracts were separated by a Fusion-RP C18 column (2 × 100 mm, 2.5 μm, phenomenex). Data acquired in selected reaction monitoring for histamine, carcinine, and β-alanine with transitions of 112/95.2, 183/95, and 90/72, respectively. Both precursor and fragment ions were collected with resolution of 0.7 FWHM. The source parameters are as follows: spray voltage: 3000 V; ion transfer tube temperature: 350°C; vaporizer temperature: 300°C; sheath gas flow rate: 40 Arb; auxiliary gas flow rate: 20 Arb; CID gas: 2.0 mTorr. Data analysis and quantification were performed using the software Xcalibur 3.0.63 (Thermo Fisher, CA). Each sample contained 50 Drosophila heads, and the mean values from five samples were calculated.