DOI: 10.25345/c5t14v170
title: MassIVE MSV000095838 - Inherent usage bias of protein quality control systems shapes tissue-specific ubiquitinome
title: We designed two multiplex TMTpro-18 experiments for gut and muscle tissue separately. For each gut sample, 330 fly guts were dissected. For each muscle sample, 100 fly thoraxes were dissected. Protein lysates were reduced with 5mM DTT and alkylated with 10mM IAM. Each sample was mixed with 12% phosphoric acid and S-Trap buffer, transferred to S-Trap column (ProtiFi, S-Trap midi columns). Samples were digested with rLys-C (Promega, V167A) at 1:100 ratio and Trypsin (Promega, V5280) at 1:50 ratio at 37C. Peptides were eluted by 0.1% FA in 50mM TEAB, and 0.1% FA in 50% ACN. Peptide was measured and dried down. To each of the TMTpro (Thermo Fisher, A52047) 0.5mg vial, 20ul of anhydrous ACN were added, then mixed with 100ug peptides and incubated 1 hr at RT. The differentially labeled samples (10ul each) were combined and the resulting volume was reduced using a SpeedVac (Savant) to less than 10ul. The dried TMT-labeled peptide mixture was resuspended in 300ul of 0.1% TFA and loaded onto the high pH fractionation cartridge (Pierce, 84868). 9 hpH RP fractions were collected by sequential elution using 300ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% ACN in 0.1% TFA. The solvents were evaporated to dryness using SpeedVac. A kit of PTMScan HS Ubiquitin/SUMO Remnant Motif (K-GG, Cell Signaling, 59322) was utilized to enrich di-Gly peptides. Each sample of 0.5mg dried peptides was resuspended with HS IAP binding buffer. Peptide solution was incubated with 10ul precleaned K-GG magnetic beads for 1 hr at 4C. For on-bead TMT labeling, 0.5mg TMTpro reagent was resuspended in 10uL of ACN and added to each sample, incubated at RT for 15min. Reactions were quenched by adding 8uL of 5% hydroxylamine to each sample at RT for 10min. All the antibody beads from each tube were combined in 1.5mL tube. 150ul 0.15% TFA was added into combined beads and incubated at RT for 10min to elute di-Gly peptides. Peptides were analyzed on Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Fisher) with a FAIMS Pro interface, equipped with a Nanospray Flex Ion Source, and coupled to a Dionex UltiMate 3000 RSCLnano System. Peptides were loaded on an Acclaim PepMap 100 C18 trap cartridge (0.3mm i.d., 5mm length; Thermo Fisher Scientific). A 75um i.d. analytical microcapillary column was packed in-house with 250mm of 1.9um ReproSil-Pur C18-AQ resin (Dr. Masch). AgileSLEEVE (Analytical Sales Products) was used to maintain column temperature at 55C. The organic solvent solutions were H2O:ACN:FA at 95:5:0.1 (volume ratio) for buffer A (pH 2.6) and 20:80:0.1 (volume ratio) for buffer B. The chromatography gradient was a 30 min column equilibration step in 2% B; a 5min ramp to reach 10% B; 90min from 10% to 40% B; 10min to reach 90% B; a 5min wash at 90% B; 0.1min to 2% B; followed by a 15min column re-equilibration step in 2% B. The nano pump flow rate was set to 0.180 ul/min. For whole proteome TMT labeled hpH fractioned samples, the Orbitrap Eclipse was set up to run the TMT-SPS-MS3 method with three FAIMS compensation voltages (CV) at -40V, -55V, and -70V. Briefly, peptides were scanned from 400-1600 m/z in the Orbitrap at 120K before MS2 fragmentation by CID at 35% NCE and detection in the ion trap set to turbo detection. Dynamic exclusion was enabled for 45s. Real time search (RTS) was performed during the ddMS2 IT CID step against a non-redundant Drosophila melanogaster sequence database downloaded from NCBI 2020-04-23 and complemented with common contaminants. Carbamidomethyl (C) and TMTpro16plex (Kn) were searched statically, while M oxidation was searched as a variable modification. Top 10 MS2 ions were selected and fragmented at 65% NCE at 50K resolution for TMT reporter ion detection. For di-Gly TMT labeled samples, the Orbitrap Eclipse was set up to run the TMT-SPS-MS3 method with 2 sets of FAIMS compensation voltages (CV) at -40V, -60V, -80V and -40V, -50V, -70V respectively. The LC/MSn dataset was processed using PD 3.0 (Thermo Fisher). MS2 spectra were searched against a non-redundant Drosophila melanogaster sequence database downloaded from NCBI 2020-04-23 complemented with common contaminants. SEQUEST-HT implemented through PD was set up as: precursor ion mass tolerance 10 ppm, fragment mass tolerance 0.3 Da, up to two missed cleavage sites. For whole proteome MS data, static modification of C (+57.021 Da), K and peptide N-termini with TMT tag (+304.2071 Da), dynamic oxidation of M (+15.995 Da) were searched. For di-Gly peptide MS data, static modification of C (+57.021 Da) and dynamic oxidation of M (+15.995 Da), K and peptide N-termini with TMT tag (+304.2071 Da), K di-Gly modification (+114.0429 Da), K TMT tag plus di-Gly modification (+418.2500) were searched. FResults were filtered to a 1% FDR at peptides levels using Percolator through PD. MS3 spectra were processed to extract intensity for each reporter ion.