The tissue was then sliced 10–20 times with a 0.2 mm minuten pin, gathered together, and covered with a 0.5 μL droplet of 1.0 M 2,5-dihydroxybenzoic acid [DHB; Sigma–Aldrich (sublimed prior to use)] prepared in 1:1 acetonitrile:water containing 2% phosphoric acid. For most extractions in acidified methanol, the extraction solvent was 30% deionized water, 65% methanol (CH3OH; HPLC-grade; Tyrosine Kinase Inhibitor Library Fisherbrand), and 5% glacial acetic acid (CH3CO2H; reagent grade; Sigma–Aldrich, ≥99%), as a %[v/v] mixture. A single eyestalk ganglion was rinsed sequentially in two 12 μL droplets of 0.75 M d-fructose

solution, placed in a 0.6 mL low retention centrifuge tube (Fisherbrand) with 50 μL of extraction solvent (smaller volumes were used when smaller tissues were analyzed), and then homogenized by one of the following methods. In early work, the tissue see more was repeatedly sliced with spring scissors; in most of the work reported in this study, tissues were ground by inserting a longer, smaller diameter polypropylene tube (0.25 mL; Fisherbrand) into the

0.60 mL tube and repeatedly twisting the tube for homogenization. In some experiments, deuterated methanol (CD3OD; 99.8% deuterated; Cambridge Isotope Laboratories, Andover, MA, USA) was substituted for the standard CH3OH in the extraction buffer (the same solvent composition was used). After tissue homogenization, the sample was sonicated for 2–5 min and centrifuged at 15k rpm for 5–15 min. The supernatant was removed from the sample and placed in another 0.6 mL tube. In early experiments, samples were delipidated prior to analysis. For delipidation, 25 μL of nanopure water was added to the supernatant along with 25 μL chloroform (NMR-grade 13CDCl3; Cambridge Isotope Laboratories) in order to remove lipids from the aqueous solution. The two layers were sonicated for 2 min and centrifuged for 10 min. The bottom organic layer was removed. Chloroform was added and the extraction was repeated two more times, but with a 5-min centrifugation. The aqueous layer

was either stored at −20 °C or concentrated to dryness in a SpeedVac vacuum concentrator (UVS400 Universal Vacuum System, Thermo Electron Corporation) Megestrol Acetate at 36 °C. Once dried, the extract was reconstituted to a total volume of 50 μL in 1:1 ACN:H2O in preparation for analysis by MALDI-FTMS or HPLC Chip–nanoESI Q-TOF MS. For some samples analyzed by MALDI-FTMS, the extracts, reconstituted in 0.1% TFA water, were desalted using C18 ZipTip pipette tips (Millipore, Billerica, MA, USA). For MALDI-FTMS analysis of extracts, 0.5 μL of the extract was mixed with 0.5 μL of DHB matrix on one face of the MALDI probe and the extract–matrix mixture was allowed to co-crystallize. For extractions in acidified acetone (85% acetone [Sigma–Aldrich, ≥99%], 13% deionized water, and 2% HCl [reagent grade; Fisherbrand], as a %[v/v] mixture, a single eyestalk ganglion was rinsed sequentially in two 12 μL droplets of 0.75 M d-fructose solution and placed in a 0.