DOI: 10.25345/c50n7q
title: MassIVE MSV000087249 - yop_TMTproj415proj476_DM_WT_dLRTS_NSUSCST5mT1hT6h
title: Protein sample preparation Proteins were extracted from 15 Drosophila melanogaster heads per sample from either CS (wild type, WT) or dLRTS (mutant) flies as follows. Heads were homogenized using a Dounce homogenizer and lysed in 200 microliter of 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1% NP-40, 0.5% Triton-X 100, EDTA-free mini protease inhibitor, 1X phosphatase inhibitor sets I and II, 0.5 microliter Bezonase (10KUN), ddH2O. Lysates were incubated at 4 degree(celsius) for 30min and then centrifuged at 10,000 rpm at 4 degree(celsius) for 10 min. Lysates (175 microliter) were transferred into new tubes and 5% were run on a 4-12% Bis-Tris gel (NuPage/Invitrogen) and silver stained. The protein extracts were treated by Methanol/Chloroform extraction followed by drying of the protein pellets. For both WT and dLRTS flies, a total of 6 conditions were analyzed: naive flies (N), starved (S), fed and stimmicroliterated (USCS), trained for 5 min (T5m), 1 hour (T1h), and 6 hours (T6h). Three biological replicates of each condition were prepared in parallel, for a total of 36 samples. Digestion and tandem mass tag labeling of total protein extracts The dried protein pellets were dissolved in 60 microliter 100 mM Tris-HCl, pH 8.5, 8 M urea, reduced in 5 mM tris(2-carboxyethyl)phosphine (Pierce), incubated at room temperature for 30 minutes, then carboxymethylated by adding choloracetamine (Sigma) to 10 mM and incubated at room temperature for 30 min in the dark. Sequential digestions with endoproteinase Lys-C and trypsin were performed at 37 degree(celsius), both overnight. The reaction was quenched with the addition of formic acid to 5%. Equal volumes of each sample (24 microliter out of 720 microliter) were aliquoted out and mixed to create a reference sample (Mix). The digested peptides were subjected to C18 solid phase extraction (Empore), followed by SpeedVac vacuum concentration to dry peptides. HPLC grade water (10.5 microliter) was added to reconstitute peptide mixture into solution. Peptide concentration was measured on 0.5 microliter of the samples using Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific). After peptide quantification, 200 mM Tetraethylammonium bromide (TEAB) was added to the peptide solution to a final concentration of 100 mM, pH 8.5. Peptides were mixed with 0.2 microliter of iRT standard peptides (Biognosys). Immediately before use, each of 0.8 mg TMT6plex or TMT11plex reagents (Thermo Fisher Scientific) was resuspended into 41 microliter of anhydrous acetonitrile and added to each of the peptide/iRT mixtures. After 1 hour, the reaction was quenched by adding 8 microliter of 50% hydroxylamine. A small amount (1 microliter) of each of the TMT-labeled peptides was analyzed by RPLC/MSMS to assess labeling efficiency, which ranged from 85 to 95%. TMT-labeled peptides were mixed at equal amount (ca. 8 ug) and dried by SpeedVac vacuum concentrator. TMT-mmicrolitertiplexed peptide mixtures were further separated into 10 to 11 fractions by high pH fractionation using Pierce cartridges (Thermo Fisher Scientific). A total of 4 different mmicrolitertiplexed batches were processed as follows: all 6 naive samples (3 biological replicates each for WT and dLRTS) were analyzed together as a TMT-6plex, while the 5 other conditions for both WT and dLRTS were analyzed in 3 TMT-11plex batches including the common mixed sample channel. Liquid chromatography mass spectrometry analysis of TMT-labeled peptide mixtures TMT-labeled and high pH fractionated peptide mixtures were analyzed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex microlitertiMate 3000 RSCLnano System. Peptides were loaded via the autosampler on an Acclaim PepMap 100 C18 trap cartridge (inner diameter (i.d.), 0.3 mm, length, 5 mm, Thermo Fisher Scientific) with the loading pump at 2 microliter/minute. A 50 micrometer i.d. analytical microcapillary column was packed in-house with 150 mm of 1.9 micrometer ReproSil-Pur C18-AQ resin (Dr. Masch). AgileSLEEVE (Analytical Sales & Products) was used to maintain column temperature at 40 degree(celsius). The organic solvent solutions were water:acetonitrile:formic acid at 95:5:0.1 (volume ratio) for buffer A (pH 2.6) and at 20:80:0.1 for buffer B. The chromatography gradient was a 50 min column equilibration step in 2% B; a 10 min ramp to reach 10% B; 180 min from 10% to 40% B; 15 min to reach 95% B; a 15 min wash at 95% B; 1 minute to 2% B; followed by a 19 min column re-equilibration step in 2% B. The nano pump flow rate was set to 150 nL/minute. The Orbitrap Fusion Lumos was set up with the SPS-MS3 method with the following settings: fmicroliterl MS1 resolution 120,000; ITMS2 isolation window 0.7 m/z, ITMS2 max ion injection time 5 ms, ITMS2 CID 35% with turbo scan, SPS ion count up to 10; FTMS3 isolation window 0.7 m/z, FTMS3 max ion injection time 105 ms, FTMS3 resolution 50,000, FTMS3 HCD energy 65%.