Dataset Information

LFQ of 3-phosphoglyceroyl lysine peptides from WT and DJ-1 KO HeLa cells

ABSTRACT: WT or DJ-1 KO HeLa cells (n=3) were lysed in 500 μL of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 x g for 15 min at 4 ºC. The supernatants were recovered and proteins (1 mg each) purified by methanol–chloroform precipitation were solubilized in 150 μL of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested overnight with 10 μg trypsin/Lys-C mix (Promega) at 37 ºC. The resulting peptide solutions were acidified with TFA, centrifuged, and used with a High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified, desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 μL/min starting at 3% B, which was linearly ramped to 32% B over 90 min, then raised to 95% B at 91 min, and held at that level until 101 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100-1700, and the ion mobility range was 0.6-1.6 V.s/cm3. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied to the m/z and ion mobility space to exclude low m/z, singly charged ions from precursor selection. The raw data were processed using FragPipe (v22.0). Database searches were performed with MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the UniProt human database (20,454 entries). Carbamidomethylation of cysteine (+57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of the protein N-terminus (+42.0106 Da); oxidation of methionine (+15.9949 Da); phosphorylation (+79.9663 Da) of serine, threonine, or tyrosine; and 3-phosphoglyceroyl (+167.9824 Da) at lysine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD063699 | JPOST Repository | Wed May 07 00:00:00 BST 2025

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	PJ1450_Ogiwara_10_DJ1_FeNTA_timsHT_DDA_Slot2-19_1_3498.d.zip	Other
	PJ1450_Ogiwara_1_WT_FeNTA_timsHT_DDA_Slot2-10_1_3480.d.zip	Other
	PJ1450_Ogiwara_2_DJ1_FeNTA_timsHT_DDA_Slot2-11_1_3482.d.zip	Other
	PJ1450_Ogiwara_5_WT_FeNTA_timsHT_DDA_Slot2-14_1_3488.d.zip	Other
	PJ1450_Ogiwara_6_DJ1_FeNTA_timsHT_DDA_Slot2-15_1_3490.d.zip	Other

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Project description:WT or DJ-1 KO HeLa cells (n=3) were lysed in 500 μL of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 x g for 15 min at 4 ºC. The supernatants were recovered and proteins (1 mg each) purified by methanol–chloroform precipitation were solubilized in 150 μL of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested overnight with 10 μg trypsin/Lys-C mix (Promega) at 37 ºC. The resulting peptide solutions were diluted 6-fold with HBS (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl), centrifuged, and used with a PTMScan Carboxymethyl/Carboxyethyl Lysine Motif kit (Cell Signaling Technology). The eluates in 0.15% TFA and 5% acetonitrile were desalted, evaporated, and re-dissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 μL/min, starting at 5% B and ramping linearly to 20% B by 40 min, then to 35% B by 60 min, followed by a rapid increase to 95% B at 61 min, where it was held until 65 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100-1700, and the ion mobility range was 0.6-1.6 V.s/cm3. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied to the m/z and ion mobility space to exclude low m/z, singly charged ions from precursor selection. The raw data were processed using FragPipe (v22.0). Database searches were performed with MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the UniProt human database (20,454 entries). Carbamidomethylation of cysteine (+57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of the protein N-terminus (+42.0106 Da); oxidation of methionine (+15.9949 Da); and carboxymethylation (+58.0055 Da) or carboxyethylation (+72.0211 Da) of lysine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.

Project description:HeLa cells (n=2) were treated with cell culture media containing 2 mM MGO or GO for 2 h and lysed in 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 x g for 15 min at 4 ºC. The supernatants were recovered and proteins (300 µg each) purified by methanol–chloroform precipitation were solubilized in 150 μL of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested overnight with 3 μg trypsin/Lys-C mix (Promega) at 37 ºC. The resulting peptide solutions were diluted 6-fold with HBS (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl), centrifuged, and used with a PTMScan Carboxymethyl/Carboxyethyl Lysine Motif kit (Cell Signaling Technology). The eluates in 0.15% TFA and 5% acetonitrile were desalted, evaporated, and re-dissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 μL/min, starting at 5% B and ramping linearly to 20% B by 40 min, then to 35% B by 60 min, followed by a rapid increase to 95% B at 61 min, where it was held until 65 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100-1700, and the ion mobility range was 0.6-1.6 V.s/cm3. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied to the m/z and ion mobility space to exclude low m/z, singly charged ions from precursor selection. The raw data were processed using FragPipe (v22.0). Database searches were performed with MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the UniProt human database (20,454 entries). Carbamidomethylation of cysteine (+57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of the protein N-terminus (+42.0106 Da); oxidation of methionine (+15.9949 Da); and carboxymethylation (+58.0055 Da) or carboxyethylation (+72.0211 Da) of lysine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.

Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:Purpose: this study is to analyze the chromatin landscape of H3R2mes in Plasmodium falciparum. Methods: In this study, H3R2me2s enrichment in the chromatin of wildtype parasite at schizont stage was analyzed by Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with next generation sequencing (CUT&Tag-seq). Synchronized WT 3D7 at 40–46 hpi schizonts were harvested and fixed with 1% formaldehyde for 1 min at room temperature, followed by lysis with 0.06% saponin. The parasite pellets were suspended in 100 μl nuclear extraction buffer (20 mM HEPES–KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1x EDTA-free Protease Inhibitor cocktail). Ten μl of activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei) and incubated for 10 min. The bead-bound nuclei were resuspended in 50 μl Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail, 0.01% digitonin, 2 mM EDTA) containing 0.5 μg of rabbit anti-H3R2me2s (Epigentek #A-3705-050) as the primary antibody or rabbit IgG (Cell Signaling Technology #2729S) serving as a control. The primary antibody was then removed, and nuclei were then incubated with 0.5 μg of the secondary antibodies in 50 μl of Digitonin150 buffer (Antibody150, no EDTA) at room temperature for 30 min. The secondary antibodies were mouse anti-rabbit IgG (Invitrogen #31213). The nuclei were washed thrice with 200 μl Digitonin150 Buffer for 5 min to remove unbound antibodies and finally resuspended in 50 μl Digitonin300 Buffer (Digitonin150 but with 300 mM NaCl). Then, 2.5 μl of CUTANA pAG-Tn5 (EpiCypher #15-1017) were added to each reaction at room temperature for 1 h. The nuclei were washed thrice for 5 min in 200 μl Digitonin300 Buffer to remove the unbound pA-Tn5. Next, the nuclei were resuspended in 50 μl Tagmentation buffer (10 mM MgCl2 in Digitonin300) and incubated at 37 °C for 1 h. The beads were washed with 50 µl TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA), mixed with 5 µL SDS release buffer (10 mM TAPS pH 8.5; 0.1% SDS) to quench tagmentation, and then incubated for 1 hr at 58ºC to release tagmented chromatin into solution. Finally, 15 µl of 0.67% Triton-X was added to each reaction to quench SDS. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by incubating with 1.3 × Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and next-generation sequencing was performed on the NextSeq 550 platform. The reads of CUT&Tag-seq from three biological replicates were mapped to the P. falciparum genome (PlasmoDB Release 39.0) by using BWA (Li and Durbin 2009) after FastQC quality control. The peak calling was finished by MACS2 version 2.2.7.1 with parameters ‘callpeak -f BAMPE’ and a q-value cutoff of 0.05. Results: The three replicates of CUT&Tag-seq using nuclei from the schizont stage showed high reproducibility, with correlation coefficients of ~0.8. Peak calling using criteria of presence in at least two of three replicates with at least 50% overlap of the peaks identified 1629 H3R2me2s signals, distributed among the 14 chromosomes. Among these peaks, 57% (927) are localized at 5’ UTR regions of 791 genes, 35% (577) at coding regions of 470 genes, and 8% (125) at 3’ UTR regions of 115 genes. The peaks are ~17.44 bp wide and ~1265 bp from the ATG sites. Comparing the transcriptome data showed genes with H3R2me2s enrichment in the 5’ UTRs showed significantly higher expression than other genes in the genome, suggesting that H3R2me2s is associated with gene activation. In addition, H3R2me2s peaks were highly co-localized with the active mark H3K9Ac, H2A.Z, and H3K4me3, further implying H3R2me2s as a euchromatin mark. Gene ontology (GO) enrichment analysis revealed a multitude of cellular pathways, including merozoite invasion, transcription, translation, stress response, cell cycle, schizogony, cell adhesion, exit from RBC, vesicle transport, signal transduction, DNA repair, and telomere organization, which are potentially regulated by this euchromatic mark. Conclusions: Collectively, H3R2me2s is a active chromatin mark and regulates invasion and other important processes in human malaria parasite.

Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.

Dataset Information

LFQ of 3-phosphoglyceroyl lysine peptides from WT and DJ-1 KO HeLa cells

Dataset's files

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