DOI: 10.25345/c5c53f54b
title: MassIVE MSV000090140 - Characteristics and molecular basis of intron retention in adult Drosophila head
title: Digestion and tandem mass tag labeling of total protein extracts The protein pellets were dissolved in 60 ul 100 mM Tris-HCl, pH8.5, 8M urea, reduced in 5mM TCEP (Pierce), incubated at RT for 30 minutes, then carboxymethylated by adding CAM (Sigma) to 10mM and incubated at RT for 30min in the dark. Sequential digestions with endoproteinase Lys-C and trypsin were performed at 37C, both overnight. The reaction was quenched with the addition of formic acid to 5%. Equal volumes of each sample (24 ul out of 720 ul) were aliquoted out and mixed to create a reference sample (Mix). The peptides were subjected to C18 SPE, followed by SpeedVac to dry peptides. HPLC grade water was added to reconstitute peptide mixture into solution. Peptide concentration was measured on 0.5 ul of the samples using Pierce Quantitative Colorimetric Peptide Assay. After peptide quantification, 200mM TEAB was added to the peptide solution to a final concentration of 100mM, pH8.5. Peptides were mixed with 0.2uL of iRT standard peptides (Biognosys). Immediately before use, each of 0.8mg TMT6plex or TMT11plex reagents was resuspended into 41uL of anhydrous acetonitrile and added to each of the peptide/iRT mixtures. After 1 hour, the reaction was quenched by adding 8uL of 50% hydroxylamine. 1 ul 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 and dried by SpeedVac. TMT peptide mixtures were further separated into 10 fractions by high pH fractionation. A total of 4 different multiplexed 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 UltiMate 3000 RSCLnano System. Peptides were loaded on an Acclaim PepMap 100 C18 trap cartridge, the loading pump at 2 uL/minute. A 50 um i.d. analytical microcapillary column was packed in-house with 150 mm of 1.9 um ReproSil-Pur C18-AQ resin (Dr. Masch). AgileSLEEVE (Analytical Sales & Products) was used to maintain column temperature at 40C. The organic solvent solutions were water:acetonitrile:formic acid at 95:5:0.1 (volume ratio) for buffer A (pH2.6) 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: full MS1 resolution 120,000; ITMS2 isolation window 0.7 m/z, ITMS2 max ion injection time 5 ms, ITMS2 CID 35%, 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%. MS data processing with kite-ProLuCID Peak list files were extracted to ms2 format with our in-house raw data processing software, RawDistiller v 0.0.1. ProLuCID v 1.3.3 was used to search the MSn dataset against a Drosophila melanogaster protein database. Mass tolerances for parent ions and fragment ions were set as 7 ppm and 800 ppm, respectively. Trypsin specificity was imposed on both ends of candidate peptides. M oxidation 15.9949 and TMT labeling of K and peptide N-termini 229.163 were set as variable modifications. A static modification with a mass of 57.0125 was added to C to account for carboxymethylation. The search engine result files were processed with the post-search processing tools, DTASelect-Contrast v 1.9, Percolator v 3.1 and Trans-Proteomics Pipeline TPP v 4.8.8 through our in-house script, swallow v. 0.0.1, which controls FDRs. FDR control was carried out by maximizing spectral counts SpC, peptides, and proteins to less than 1% FDRs when ProLuCID Xcorr and DeltCN and the DTASelect r minimum spectra required for a positively identified peptide and p minimum peptides required for a positively identified protein increased. A script module was added into NSAF7 to extract intensity for each reporter ion in MS3 spectra via Xcalibur. Proteins were quantified by summing reporter ion intensities across all matching PSMs. The PSM qualitative and quantitative reports were generated with our in-house software, NSAF7 v 0.0.1. The dIAF is calculated with proteins distributive TMT intensity/protein length. The xdNIAF is calculated with dIAF/sumdIAF*1013. The NIAFw is the cubic root of xdNIAF. The xnTPMM for mRNA dataset is calculated with TPM/sumTPM*1012. The TPMMw is the cubic root of xnTPMM.