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A CRISPR-Cas13d screen uncovers Bckdk as a regulator of the maternal-to-zygotic transition in teleosts

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Danio Rerio (ncbitaxon:7955)

SUBMITTER: Laurence Florens

PROVIDER: MSV000094733 | MassIVE | Thu May 09 19:55:00 BST 2024

SECONDARY ACCESSION(S): PXD052161

REPOSITORIES: MassIVE

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Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.

Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.

Project description:Proteomic analysis Epilepsy was induced using perforant pathway stimulation (PPS) in rats as described (Norwood BA, et al. (2010) Classic hippocampal sclerosis and hippocampal-onset epilepsy produced by a single "cryptic" episode of focal hippocampal excitation in awake rats. J Comp 803 Neurol 518(16):3381-3407). Rats were killed under deep anesthesia (xylazine+ketamine) by transcardial perfusion with ice-cold 0.9 % NaCl solution at the following time points: 1 h, 24 h, 72 h, 10 d and 16 d after PPS (epileptogenesis); within 1 d of first spontaneous seizure (early epilepsy); 1 month after first spontaneous seizure (chronic epilepsy). Control rats were killed on day 17 after surgery (corresponding to day 10 after PPS). Hippocampi were removed and snap frozen at -80°C. Frozen rat hippocampi were resuspended in 200 ul lysis buffer (6M GdmCl, 10mM TCEP, Complete protease inhibitor cocktail, PhosSTOP inhibitor, 100 mM TEAB, benzonase) and dounced 30 times on ice. Samples were boiled for 5 min, sonicated on ice for 2min and centrifuged at 13000 rpm for 3 min. The supernatant was added chloroacetamide to a final concentration of 20 mM followed by incubation in the dark for 30 min at room temperature (RT). Proteins were digested with LysC for 30 min at RT and then diluting 10x— using 100 mM TEAB followed by adding trypsin and incubating overnight at 37°C with shaking. LysC and trypsin were added in a LysC/trypsin:protein ratio of 1:100. Samples were centrifuged at 13000 rpm for 5 min and from the supernatant a volume corresponding to 50 ug peptide was transferred to a new eppendorff tube. Sets of 6 samples were labelled using the TMTsixplex isobaric label reagent. (ThermoFisher Scientific). The labeling reagent was prepared using the manufactures instructions to first equilibrate to RT, then dissolving the labeling reagent in 41 ul anhydrous acetonitrile. The labeling reagent was added to each sample and incubated for 1 hour at RT. The reaction was quenched by incubating with 0.76 M lysine for 15 min. A small amount was taken from each sample, mixed and tested by mass spectrometry for labeling efficiency and mixing ratio. Remaining samples were mixed in a 1:1 ratio. The Stage-tips for the high-pH reverse phase fractionation were prepared by placing first two C18 disks in a p200 pipette tip, then adding a slurry of C18 beads (ReproSil-Pur, 3 um) in methanol and adding one additional C18 disk in the top. The column was activated and equilibrated by washing one time in 150 ul buffer B (50% buffer A, 50% ACN) and then two times in 150 ul buffer A (20mM Ammonium hydroxide, pH 10). The labeled peptide sample was adjusted to pH 10 using buffer A and then loaded onto the activated and equilibrated stage-tip by spinning the sample through the column and collecting 26 the flow-through. The column was washed 2 times with 150 ul buffer 585 A and the washes were collected and combined with the flow-through. Next, peptides were eluted stepwise in 17 fractions by spinning 150 ul of buffer A containing increasing amounts of ACN for each step (3.5%, 4.5%, 6%, 8%, 10%, 10.5%, 11.5%, 13.5%, 15%, 16%, 17.5%, 18.5%, 19.5%, 21%, 27%, 50%, 80%) through the column. After collection, the liquid was evaporated completely in a Concentrator Plus (Eppendorf) and prepared for MS by resuspending in buffer A* (2% ACN, 0.1% TFA). Samples were analyzed using an Easy-nLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source (Proxeon/Thermo Scientific). The peptides were eluted into the mass spectrometer during a chromatographic separation from a fused silica column packed in-house with 3 um C18 beads (Reprosil, Dr. Maisch) using a 120 min gradient of buffer B (80% ACN, 0.5% acetic acid) with a flow rate of 250 nL/min. The Q Exactive HF was operated in positive ion mode with a top 12 data-dependent acquisition method where ions were fragmented by higher-energy collisional dissociation (HCD) using a normalized collisional energy (NCE)/ stepped NCE of 28 and 32. The resolution was set to 60,000 (at 400m/z), with a scan range of 300-1700 m/z and an AGC target of 3e6 for the MS survey. MS/MS was performed at a scan range of 100 - 2000 m/z using a resolution of 30,000 (at 400 m/z), an AGC target of 1e5, an intensity threshold of 1e5 and an isolation window of 1.2 m/z. Further parameters included an exclusion time of 45 sec and a maximum injection time for survey and MS/MS of 15 ms and 45 ms respectively.

Project description:The experiment consists of M.hyp 232 cultures digests analyzed on two mass specs, LTQ Velos Pro and LTQ FT Ultra LTQ Velos Pro: Six cell culture replicates of M.hyo 232 were hydrophobically separated using tree detergents: Digitonin, Tween, and SDS. Each francion, including the insoluble pellet, was trypsin digested and then cleaned using an SCX trap follwed by a RP trap to remove detergents. Each fraction was sepated using a Dionex U3000 splitless nanoflow system operating at 333 nl per minute using a gradient of 2% ACN to 50% ACN in 4 hours. Eluate was analyzed using an LTQ Velos Pro mass spectrometer with 20 MS/MS scans of the 20 most intense peaks from each MS scan. Dynamic exclusion was enable for 3 minutes for each m/z with a repeat count of 1. LTQ FT Ultra: Two cell pellets for high resolution analysis were lysed and trypsin digested. Digested peptides were dried, resuspended in 20 mM KH2PO4, 20% ACN, pH 3 (Buffer A) in 2.5 µL and transferred to low retention vials in preparation for separation using an Ultimate 3000 configured for 2D-LC. Each sample was loaded at 15 µl/min onto an SCX microtrap for the first dimension of separation, involving SCX steps of Buffer A + 0, 5, 10, 15, 20, 25, 30, 40, 50, 100, 250, 500, and 1000 mM KCl. For the second dimension of separation, each eluted salt step was desalted with an inline peptide microtrap with 2% ACN, 0.1% FA at 5 µl/min. Once desalted, the microtrap was switched into line with a fritless nano column (75µm x ~10cm) containing C18 media (5µ, 200 Å Magic, Michrom). Peptides were eluted using a gradient of 2% to 36% ACN, 0.1% FA at 350 nl/min over 60 min and electrospray ionized for analysis using an LTQ FT Ultra mass spectrometer. A survey scan m/z 350-1750 was acquired in the FT ICR cell (Resolution = 100,000 at m/z 400, with an accumulation target value of 1,000,000 ions). Up to the 6 most abundant ions (>3,000 counts) with charge states > +2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/ MS were dynamically excluded for 30 seconds. Analysis: X!tandem searches were performed using the Mycoplasma hyopneumoniae strain 232 reference protein set from NCBI. The only difference between the searches for the LTQ Velos and LTQ FT was the precursor mass tolenance being set to +-1500ppm and +-24ppm respectively. Decoy searches were performed and the data filtered at e-value <= 0.01 with single peptide proteins discarded. These results are included in the submission as two tab-delimited text files.

Project description:Serum samples were inactivated and sterilized at 56 ℃ for 30 min and processed as previous study with some modifications (Shen et al., 2020). Briefly, 4 μL serum from each sample was depleted using High Select Top-14 Abundant Protein Depletion MiniSpin Columns (Thermo Fisher Scientific, San Jose, USA). Then the elutes were denatured, reduced and alkylated to derive protein lysates. The solutions were diluted with 200 μL 100 mM triethylammonium bicarbonate (TEAB) followed by a double-step trypsinization (enzyme-to-substrate ratio kept at 1:40 in each step). 10% trifluoroacetic acid (TFA) was added to each sample to quench the enzymatic reaction. Digested peptides were cleaned-up with SOLAμ (Thermo Fisher Scientific, San Jose, USA) according to the manufacturer’s instructions and labeled by TMTpro 16 plex reagents (Thermo Fisher Scientific, San Jose, USA). 660 samples in total (633 serum samples and 27 technical replicates) were divided into 44 batches for labelling experiment, each batch containing fifteen samples and one pool. The labeled samples in each batch were combined and fractionated. Each sample was separated into 30 fractions and then consolidated into 10 fractions. Dried and re-dissolved fractions with 2% acetonitrile (ACN)/0.1% formic acid (FA) of MS grade. The re-dissolved peptides were analyzed with a U3000 HPLC system coupled to a Orbitrap Exploris 480 (Thermo Fisher Scientific, Bremen, Germany) in data-dependent acquisition (DDA) mode combined to a front-end high-field asymmetric waveform ion mobility spectrometry (FAIMS). Peptides of each fraction were loaded onto a pre-column (3 μm, 100 Å, 20 mm*75 mm i.d.) and separated with a 75 min LC gradient at a flow rate of 300 nL/min (analytical column: 1.9 mm, 120 Å, 150 mm*75 mm i.d.; Buffer A: 2% ACN, 98% H2O containing 0.1% FA; Buffer B: 98% ACN, 2% H2O containing 0.1% FA). FAIMS was operated at two different CV, -48 V and -68 V, respectively. For MS acquisition, RF level was set at 50% and ion transfer tube temperature at 320 °C. The turbo-TMT mode was enabled. Scan range of MS1 was 350–1800 m/z. Resolutions were set to 60000 for MS1 and 30000 for MS2. Normalized AGC target was set at 300% for MS1 and 200% for MS2. The maximum injection time was set as 50 ms for MS1 and 86 ms for MS2. Dynamic exclusion was on and mass tolerance was set to ±10 ppm. Intensity threshold was set at 20000 for MS1, and HCD collision energy was fixed to 38%. MS/MS data was recorded in centroid mode. Isolation window was set to 0.7 m/z.

Project description:Lubricin or proteoglycan-4 (PRG-4) is a mucinous glycoprotein that lubricates the cartilage and maintains normal tissue function and cell homeostasis. Altered O- glycoproteforms of lubricin have been found in osteoarthritis (OA) synovial fluid (SF), which could ostensibly be used as a biomarker to diagnose early onset OA. However, SF is invasive to obtain and generally would not be surveyed on otherwise healthy individuals. Thus, a plasma-based OA screening tool focused on lubricin glycosylation could serve as an improved biomarker. In this report, we used glycomics and glycoproteomics to identify glycoproteoforms of OA lubricin in SF and plasma. We obtained near-complete sequence coverage of the mucin domain of lubricin and its glycosylation using matched SF/plasma from OA patients (N=5). In SF we observed a spectrum of O-glycans on lubricin ranging from the single GalNAc monosaccharide up to branched pentasaccharide and low in sialylation compared lubricin in plasma, decorated with sialylated Gal1-3GalNAc. We also detected splice variant-specific peptides from lubricin non-glycosylated region showing that the longest splice form of lubricin was exclusively present in SF, while in addition shorter splice variants were also present in plasma.Following elution, the mucin-enriched plasma or synovial fluid was subjected to reduction, alkylation, and proteolytic digestion. To begin, dithiothreitol (DTT, Sigma) was added to a concentration of 2 mM and reacted at 65 °C for 20 min followed by alkylation in 5 mM iodoacetamide (IAA, sigma) for 15 min in the dark at RT. Subsequently, mucinase SmE was added at an enzyme:substrate (E:S) ratio of 1:20 and allowed to react overnight at 37 °C. At this point, the samples were split into two aliquots, one containing 90% and the other containing the remaining 10%. The former represented the “mucinase-only” digest that did not undergo any further proteolysis. The latter was subjected to an additional digestion with trypsin at a 1:50 E:S ratio for 6 hours at 37 °C. All reactions were quenched by adding 1 µL of formic acid (Thermo Scientific) and diluted to a volume of 200 µL prior to desalting. Addition of formic acid also caused sodium deoxycholate to precipitate out of solution, and the resulting supernatant was transferred to a new tube before desalting. Desalting was performed using 10 mg Strata-X 33 µm polymeric reversed phase SPE columns (Phenomenex). Each column was activated using 500 µL acetonitrile (ACN) (Honeywell) followed by of 500 µL of 0.1% formic acid, 500 µL of 0.1% formic acid in 40% ACN, and equilibration with two additions of 500 µL of 0.1% formic acid. After equilibration, the samples were added to the column and rinsed twice with 200 µL of 0.1% formic acid. The columns were transferred to a 1.5 mL tube for elution by two additions of 150 µL of 0.1% formic acid in 40% ACN. The eluent was then dried using a vacuum concentrator (LabConco) prior to reconstitution in 10 µL of 0.1% formic acid.

Project description:Cyanophora paradoxa muroplasts were isolated on a Percoll gradient. Muroplasts were fractionated into soluble (stroma), envelope, and thylakoid proteins. Protein samples from envelope membranes, thylakoids, stroma and total muroplasts were separated by 12% SDS-PAGE. Each Protein lanewas cut into 10 slices of variable size to match similar protein amounts. The gel slices were washed and diced to 1mm3 cubes. Gel pieces were destained by two consecutive washes with a 50:50 mixture of 200 mM ammonium bicarbonate and acetonitrile (ACN) for 20 min at 37°C. Proteins were reduced with 10mM DTT for 45 min at 56°C, followed by alkylation with 55mM iodoacetamide for 45 min at room temperature in the dark. After reduction and alkylation, gel pieces were washed twice with 100 mM ammonium bicarbonate, dehydrated with 100% ACN for 10 min, and dried in a SpeedVac. Gel pieces were fully covered in 50 mM ammonium bicarbonate containing 10ng/ml trypsin and rehydrated at 4°C. The samples were incubated overnight at 37°C and peptides in the supernatant were pooled after successive extraction steps using 50 mM ammonium bicarbonate, 100% ACN and 2.5% formic acid (FA), 50% ACN. The samples were dried in a SpeedVac and reconstituted in 5% ACN, 0.1% FA prior to MS analysis. Each of the samples were analysed in duplicates. Tryptic peptides of the thylakoid, envelope and stroma fractions were loaded onto a fritless 100 μm capillary packed in-house with 10cm of ProntoSIL C18 ace-EPS, 3µm. A gradient of 5-80% (v/v) ACN in 0.1% (v/v) FA was passed over the column with a duration of 115 min. The eluted peptides were directly electrosprayed into the LTQ orbitrap XL MS at a flow rate of 250 nl min-1 and a spray voltage of 1.3 kV. The instrument was operated in a 4-step data-dependent positive mode to identify the top three most abundant ions in a high-resolution MS scan (400 to 2000 m/z), which were then selected for fragmentation. MudPIT analyses were performed on the muroplast samples using an in-house packed analytical column consisting of 10 cm ProntoSIL C18 ace-EPS, 3µm (Bischoff) and 2 cm of a strong cationic exchange (SCX) material, 3µm in combination with an 6-step salt pulse gradient (0, 50, 100, 150, 200 and 500 mM ammonium acetate). Each salt-step peptides were eluted from the SCX to the C18-phase of the analytical column and eluated with a 130- min reverse-phase solvent gradient (5–80% ACN containing 0.1% FA). The eluate was directly electrosprayed into the LTQ orbitrap XL MS which was operated as described before. All MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search the C. paradoxa nuclear and muroplast protein database with a total of 32,286 entries, assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the C. paradoxa database also assuming trypsin. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0,80 Da and a parent ion tolerance of 10,0 ppm. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in Sequest and X! Tandem as variable modifications. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95,0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99,0% probability and contained at least 2 identified peptides.

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A CRISPR-Cas13d screen uncovers Bckdk as a regulator of the maternal-to-zygotic transition in teleosts

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