Ammonium acetate buffer

On the day of analysis, buffer was exchanged to 100 mM ammonium acetate buffer (Fisher Scientific, Loughborough, UK) of specified pH, using micro Bio-Spin Chromatography columns (Micro Bio-Spin 6 Columns, Tris) following the instructions specified by the manufacturer. The desalting procedure was performed four to five times to achieve desired sample quality. pH of the buffer was adjusted with hydrochloric acid or ammonia supplied by VWR International Ltd (UK). Solution pH readings were taken using a pH meter (Jenway 3305). High purity water was obtained from an Arium 611 water purification unit (Sartorius, Göttingen, Germany) fitted with a 0.2 μm filter. Charge reduction experiments were carried out by addition of 10% (v) triethylamine acetate buffer (TEAA) (Fluka, Steinheim, Switzerland) of ethylenediammonium diacetate (EDDA) prior to MS analysis.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to confirm drug uptake in our control and transgenic fly cohorts (see Supplementary Figure S1). Tissues were homogenized in 5 mM ammonium acetate buffer (Fisher Scientific, Waltham MA, USA). An aliquot of 50 μL was used for analysis. Calibration standards, blanks, and quality controls (QCs) were prepared by spiking naïve homogenate (100 µL) with the appropriate amount of lisinopril with concentrations in the tissue homogenate ranging from 5 to 20,000 ng/mL. Samples and standards were fortified with an internal standard,5 µL of 100 ng/mL Enalaprat (Cayman Chemical, Ann Arbor, MI, USA) in DI water, and extracted with 0.5 mL 90:10 methanol: acetone (Fisher Scientific, Waltham, MA, USA). Samples were vortexed well, centrifuged and the supernatant was evaporated under nitrogen at 50 °C. Samples were redissolved in 200 µL DI water and transferred to limited-volume autosampler vials for analysis by LC–MS/MS. Detection was accomplished using an Applied BioSystems 4000 QTRAP (Applied Biosystems, Foster City, CA, USA) triple quadrupole mass spectrometer equipped with an electrospray ionization source operated at a potential of 5 kV at 450 °C operating in the MRM mode. Data were collected using Analyst 1.6.2 (Applied Biosystems, Foster City, CA, USA) and normalized to protein concentration.