Dataset Information

Proteomic analysis of SovN-associated proteins immunopurified with an anti-FLAG antibody

ABSTRACT: Immunoprecipitated proteins on beads were washed once and resuspended in 50 mM ammonium bicarbonate, followed by the addition of 200 ng trypsin/Lys-C mix and an overnight incubation at 37ºC. The digests were reduced, alkylated, acidified, and desalted with GL-Tip SDB. Then, the eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75-µm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 min followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometers were operated in data-dependent acquisition mode. Raw data were directly analyzed against the UniProt database restricted to Drosophila melanogaster using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Kensaku Murano

PROVIDER: PXD068509 | JPOST Repository | Fri Sep 19 00:00:00 BST 2025

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	146_Murano_1_C36_Dox_minus_FLAG_IP_FusionIT.raw	Raw
	148_Murano_2_C36_Dox_plus_FLAG_IP_FusionIT.raw	Raw
	150_Murano_3_C47_Dox_minus_FLAG_IP_FusionIT.raw	Raw
	152_Murano_4_C47_Dox_plus_FLAG_IP_FusionIT.raw	Raw
	230827_Murano_SovN_FLAG_IP_FusionIT.xlsx	Xlsx

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Publications

The Piwi-piRNA complex initiates transposon silencing via transcription termination factors PNUTS and Senataxin.

Wu Yaning Y Imai Rin R Kosako Hidetaka H Nakahara Toshiya T Solberg Therese T Siomi Haruhiko H Murano Kensaku K

Molecular cell 20251029 21

Transcriptional silencing of transposable elements (TEs) is guided by Piwi and PIWI-interacting RNAs (piRNAs) through the SFiNX complex to induce heterochromatin formation. We previously reported that members of the SFiNX complex can induce TE silencing prior to heterochromatin formation, suggesting that Piwi-mediated silencing can be separated into two steps: initiation and maintenance. While many studies have investigated the heterochromatin-dependent maintenance step of TE silencing, the mech ...[more]

PMID: 41167190

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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: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:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.

Project description:Anti-Flag immunoprecipitated 3×Flag-SUMO-T86K proteins on beads were washed once and suspended in 50 mM ammonium bicarbonate, followed by the addition of 1 µg Lys-C and an overnight incubation at 37ºC. The digests were reduced, alkylated, diluted 10-fold with HBS (50 mM HEPES-NaOH pH7.5, 150 mM NaCl), and incubated with PTMScan HS K-ε-GG Remnant Magnetic Immunoaffinity Beads (Cell Signaling Technology) for 2 h at 4ºC. After washing with HBS three times, peptides were eluted with 200 µL of 0.1% TFA and 60% ACN. The eluates were evaporated, dissolved in 0.1% TFA, and desalted with GL-Tip SDB. After evaporation, the resultant peptides were dissolved in 0.1% TFA and 3% ACN and analyzed by an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer. The peptides were separated on a 75-µm inner diameter × 150 mm C18 reversed-phase column with a linear 4-32% ACN gradient for 0-60 min followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 seconds. Raw data were directly analyzed against the UniProt database restricted to Drosophila melanogaster using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters were as follows: (a) Lys-C as an enzyme with up to 2 missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; and (d) acetylation of protein N-terminus, oxidation of methionine, carbamidomethylation or N-ethylmaleimidation of cysteine, and di-glycine modification of lysine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Anti-Flag immunoprecipitated 3×Flag-SUMO-T86R proteins on beads were washed once and suspended in 50 mM ammonium bicarbonate, followed by the addition of 1 µg trypsin/Lys-C mix and an overnight incubation at 37ºC. The digests were reduced, alkylated, diluted 10-fold with HBS (50 mM HEPES-NaOH pH7.5, 150 mM NaCl), and incubated with PTMScan HS K-ε-GG Remnant Magnetic Immunoaffinity Beads (Cell Signaling Technology) for 2 h at 4ºC. After washing with HBS three times, peptides were eluted with 200 µL of 0.1% TFA and 60% ACN. The eluates were evaporated, dissolved in 0.1% TFA, and desalted with GL-Tip SDB. After evaporation, the resultant peptides were dissolved in 0.1% TFA and 3% ACN and analyzed by an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer. The peptides were separated on a 75-µm inner diameter × 150 mm C18 reversed-phase column with a linear 4-32% ACN gradient for 0-60 min followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 seconds. Raw data were directly analyzed against the UniProt database restricted to Drosophila melanogaster using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters were as follows: (a) trypsin as an enzyme with up to 2 missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; and (d) acetylation of protein N-terminus, oxidation of methionine, carbamidomethylation or N-ethylmaleimidation of cysteine, and di-glycine modification of lysine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).