DOI: 10.25345/c5pz51z07
title: MassIVE MSV000094733 - A CRISPR-Cas13d screen uncovers Bckdk as a regulator of the maternal-to-zygotic transition in teleosts
title: Samples were reduced by 5mM TECP at rt for 30min and carboxymethylated by 10mM 2-chloroacetamide for 30min in dark at rt. Pre-chilled acetone was added to precipitate proteins overnight. The precipitated proteins were collected and dissolved in 100ul of 50mM TEAB, MS grade trypsin (Promega Gold) was added at 1:40 w/w at 37C overnight. Peptide amounts were measured by fluorometric peptide assay (Pierce). 20ul of anhydrous acetonitrile (ACN) were added into each 0.5mg TMTpro 16plex reagents (Thermo Scientific). For each sample, 20ug peptides were added into TMT vials. The 4 replicate samples for the WT non-injection, WT cas13-injection, BCKDK cas13-injection, were labeled with TMTpro respectively. The 12 differentially labeled samples were combined and concentrated by SpeedVac concentrator (Savant) to less than 10ul. High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific) was used for phosphopeptide enrichment. Lyophilized TMT labeled peptides were suspended in 200ul of binding buffer and loaded onto equilibrated Fe-NTA spin column. After 30min incubation, the supernatant was collected through centrifugation. Phosphopeptides were eluted by 100ul of elution buffer. Both supernatant and eluate were dried immediately to less than 10ul. The dried supernatant TMT-labeled peptide mixture was resuspended in 300ul of 0.1% trifluoroacetic acid (TFA) and loaded onto the high pH fractionation cartridge (Pierce). 8 hpH RP fractions were collected by sequential elution using 300ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50% in 0.1% TFA. The solvents were evaporated to dryness using SpeedVac. Dried samples were resolubilized in buffer A (5% ACN in 0.1% formic acid, FA) before LC-MS analysis. TMT-labeled peptides were analyzed on Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Scientific) with a FAIMS Pro interface, coupled to a Dionex UltiMate 3000 RSCLnano System. Peptides (hpH RP fraction) were loaded on an Acclaim PepMap 100 C18 trap cartridge. A 75 um i.d. analytical capillary column was packed in-house with 250mm of 1.9um ReproSil-Pur C18-AQ resin (Dr. Masch). The organic solvent solutions were H2O:ACN:FA at 95:5:0.1 (v:v:v) for buffer A (pH 2.6) and 20:80:0.1 for buffer B. The chromatography gradient was a 30min equilibration step in 2%B; a 5-min 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 0.180ul/min. The Orbitrap Eclipse was set up to run the TMT-SPS-MS3 method with three FAIMS. Briefly, peptides were scanned from 400-1600 m/z in the Orbitrap at 120K and fragmented by CID at 35% and detected in ion trap. The supernatant from phosphorylation peptide enrichment was analyzed with real time search (RTS). During data acquisition, ddMS2 spectra was searched against a non-redundant Danio rerio sequence database (UniProt 2021-03) with common contaminants. Carbamidomethyl (C) and TMTpro16plex (Kn) were searched statically, while oxidation (M) was searched as a variable modification. TMT reporter ions were detected by MS3. The MS dataset was processed using Proteome Discoverer 2.4 (Thermo Fisher Scientific). MS/MS spectra were searched against a Danio rerio protein database with common contaminants. SEQUEST-HT implemented through PD was set up as: precursor ion mass tolerance 10 ppm, fragment mass tolerance 0.6 Dalton, up to two missed cleavage sites, static modification of C(+57.021), and K and peptide N-termini with TMT tag(+304.2071) and dynamic oxidation of M(+15.995), and phosphorylation of S, T, Y(+79.9663). Leucine and isoleucine (Sigma-Aldrich), 13C6-Leucine (Cambridge Isotopes) and samples were derivatized by dansyl chloride (Sigma-Aldrich). Dried samples and amino acids standards were dissolved in pH9.5 250mM sodium bicarbonate buffer, dansyl chloride was added and incubated at room temperature for 1hr respectively. Derivatized 13C6-Leucine was spiked into derivatized Leucine respectively, as well as each derivatized sample before LC/MS analysis. Amino acid samples were analyzed on a Q-Exactive Plus Mass Spectrometer (Thermo Scientific) equipped with a Nanospray Flex Ion Source and coupled to a Dionex UltiMate 3000 RSCLnano System. A 75um i.d. analytical capillary column was packed in-house with 100mm of 1.9um ReproSil-Pur C18-AQ resin (Dr. Masch). The UPLC solutions were 0.1% FA in H2O for buffer A and 0.1% FA in ACN for buffer B. The chromatography gradient as a 5min loading time at 20%B, 23min from 20% to 35%B; 2min to reach 100%B; and 10min washing at 100%B. The nano pump flow rate was set to 0.5ul/min. The Q-Exactive was set up to run PRM method with inclusion list as 371.1736 m/z and 365.1535 m/z. Each standard curve concentration points and each sample were analyzed in triplicates. The peak area ratio between leucine and 13C6-leucine was used to quantify the leucine amount in each sample.