GNPS - Brittonodoxa subpinnata soluble extract
Ontology highlight
ABSTRACT:
INSTRUMENT(S): HPLC-MS ESI low resolution (Thermo-Fischer)
ORGANISM(S): Brittonodoxa Subpinnata
SUBMITTER: 
Wilton Ricardo Sala-Carvalho
PROVIDER: MSV000093497 | GNPS | Sat Nov 25 14:04:00 GMT 2023
REPOSITORIES: GNPS
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2023-11-25 | MSV000093498 | MassIVE
Project description:3 µl of each human serum sample was added to 40 µl lysis buffer containing 5% sodium dodecyl sulfate (SDS) and 50 mM triethylammonium bicarbonate (TEAB), pH 8.5. Proteins were reduced by addition of 15 mM dithiothreitol and incubation for 30 minutes at 55˚C and then alkylated by addition of 30 mM iodoacetamide and incubation for 15 minutes at RT in the dark. Phosphoric acid was added to a final concentration of 1.2% and subsequently samples were diluted 7-fold with binding buffer containing 90% methanol in 100 mM TEAB, pH 7.55. The samples were loaded on the 96-well S-Trap™ plate (Protifi), placed on top of a deepwell plate, and centrifuged for 2 min at 1,500 x g at RT. After protein binding, the S-trap™ plate was washed three times by adding 200 µl binding buffer and centrifugation for 2 min at 1,500 x g at room temperature. A new deepwell receiver plate was placed below the 96-well S-Trap™ plate and 50 mM TEAB containing trypsin (1/100, w/w) was added for digestion overnight at 37°C. Using centrifugation for 2 min at 1,500 x g, peptides were eluted in three times, first with 80 µl 50 mM TEAB, then with 80 µl 0.2% formic acid (FA) in water and finally with 80 µl 0.2% FA in water/acetonitrile (ACN) (50/50, v/v). Eluted peptides were dried completely by vacuum centrifugation. Dried peptides were redissolved in 100 µl 0.1% FA, the peptide concentration was determined on a Lunatic spectrophotometer (Unchained Labs) and was adjusted to 0.015 µg/µl with 0.1% FA1. iRT peptides (Biognosys, P/N Ki-3002-1) were added to all samples according to the manufacturer’s instructions. Then, 300 ng of each sample was loaded on Evotips (Evosep, P/N EV2003) according to the manufacturer’s instructions with a substitution of the wash step after sample loading by two wash steps with 80 µl 0.1% FA. All loaded Evotips were stored in 0.1% FA at 4°C until LC-MS/MS analysis was started.
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Project description:To further study the transcriptome of THP-1 human monocytes after exposure to S-Nitrosoglutathione (GSNO), we investigate whole genome microarray expression to identify genes regulated by exposure or not to GSNO. To further study the transcriptome of THP-1 human monocytes after exposure for 4 h to 50 ug / mL of S-Nitrosoglutathione-loaded polymeric Eudragit RL nanoparticles (GSNO-loaded ENP), we investigate whole genome microarray expression to identify genes regulated by exposure or not to 50 ug / mL of GSNO-loaded ENP. To further study the transcriptome of THP-1 human monocytes after exposure for 4 h to 200 ug / mL of empty polymeric Eudragit RL nanoparticles (empty ENP), we investigate whole genome microarray expression to identify genes regulated by exposure or not to 200 ug / mL of empty ENP. To further study the transcriptome of THP-1 human monocytes after exposure for 24 h to 50 ug / mL of S-Nitrosoglutathione-loaded polymeric Eudragit RL nanoparticles (GSNO-loaded ENP), we investigate whole genome microarray expression to identify genes regulated by exposure or not to 50 ug / mL of GSNO-loaded ENP. To further study the transcriptome of THP-1 human monocytes after exposure for 24 h to 50 ug / mL of empty polymeric Eudragit RL nanoparticles (empty ENP), we investigate whole genome microarray expression to identify genes regulated by exposure or not to 50 ug / mL of empty ENP. To further study the transcriptome of THP-1 human monocytes after exposure for 4 h to 50 ug / mL of empty polymeric Eudragit RL nanoparticles (empty ENP), we investigate whole genome microarray expression to identify genes regulated by exposure or not to 50 ug / mL of empty ENP. To further study the transcriptome of THP-1 human monocytes after exposure for 4 h to 200 ug / mL of S-Nitrosoglutathione-loaded polymeric Eudragit RL nanoparticles (GSNO-loaded ENP), we investigate whole genome microarray expression to identify genes regulated by exposure or not to 200 ug / mL of GSNO-loaded ENP.
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Project description:Human Umbilical Vein Endothelial Cells pooled from several donors, were purchased from Lonza and cultured in FBS reduced Endopan 3 media. HUVEC cells of passages 5-10 were used for experiments. MPO treatment was performed with protein purified from human blood (Planta) and at 1 μg/ml final concentration. Immunoprecipitation was performed from isolated nuclei (after 8 hours MPO treatment), non-treated cells were used as negative control. For immunoprecipitation antibody against MPO (Calbiochem, 475915) was used. Cell nuclei were isolated by incubating cells for 15 min on ice in NIB buffer (15 mM Tris-HCL pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM sucrose) containing 0.3% NP-40. Nuclei were pelleted for 5 min 800 × g at 4 °C, washed twice in the same buffer, lysed for 10 min on ice in IP buffer (150 mM LiCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% Empigen) freshly supplemented with 2 mM sodium vanadate, 1× protease inhibitor cocktail (Roche), PMSF (10 µl), 0,5 mM DTT, and 50 units Benzonase per ml of IP buffer, before preclearing cell debris by centrifugation at >15,000 × g at 4 °C. Finally, 1 mg of the lysate was incubated with anti-MPO antisera overnight at 4 °C. Magnetic beads (Active Motif) were then washed once with 1× PBS-Tween and combined with the antibody-lysate mixture. Following a 2-h incubation at 4 °C, beads were separated on a magnetic rack and washed 5×, 5 min each in wash buffer (150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl pH 7.5, 0.5% NP-40) and another two times in wash buffer without NP-40. Captured proteins were predigested and eluted from the beads using digestion buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 1 mM DTT) supplemented with trypsin and eluted from the beads with elution buffer (2 M Urea, 50 mM Tris-HCl pH 7.5, 5 mM chloroacetamide) supplemented with trypsin and LysC, before subjected to mass-spectrometry on a Q-Exactive Plus Orbitrap platform coupled to an EASY nLC (Thermo Scientific). Peptides were loaded in solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm length, 75 µm I.D., filled with 2.7 µm Poroshell EC120 C1; Agilent)
2024-05-23 | PXD033079 | Pride
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.
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Project description:Pretreated human follicular fluid was prepared for targeted metabolomics experiments. For this purpose, 12-point standard calibration curves in the respective range with reference material were measured, and the limit of detection (LOD) and limit of quantification (LOQ) for each neurotransmitter under investigation were calculated. LC-MS/MS was conducted by an ultra-performance liquid chromatography method using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm, Waters, USA). The Waters acquisition UPLC was coupled to an API 4000 triple quadrupole mass spectrometer (AB SCIEX, USA). The mobile phase A buffer was 10% methanol in water (containing 0.1% formic acid), and the mobile phase B buffer was 50% methanol in water (containing 0.1% formic acid). The LC gradient elution was at a flow rate of 0.3 mL/min at 0-8.0 min and 0.4 ml/min at 8.0-17.5 min, then 10%-30% B at 0-6.5 min; 30%-100% B at 6.5-7 min; 100% B at 7-14min; and 100%-10% B at 14-17.5min. The injection volume was 5 µl and the column temperature was set to 40 °C. Twenty-three metabolites (Ach: Acetylcholine chloride; Gln: Glutamine; Glu: Glutamate; His: L-Histidine; NE: norepinephrine; Tyr:Tyrosine; Trp:Tryptophan; Kyn: Kynurenine; GABA: 4-Aminobutyric acid;HisA:Histamine; PA:Picolinic acid;TyrA:Tyramine;DA:Hydroxytyramine hydrochloride; TrpA:Tryptamine;5-HT:Serotonin hydrochloride;E:Adrenaline hydrochloride;KynA:Kynurenic acid;5-HIAA:5-Hydroxyindole-3-acetic acid;DOPA:Levodopa;XA:Xanthurenic acid;VWA:Vanillymandelic Acid; 5-HTTP:5-Hydroxytryptophan;MT:Melatonine)were simultaneously reported in LC-MS/MS multiple reaction monitoring (MRM) mode. Data analysis was performed with Analyst 1.6 software.
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Project description:The plant leaves were incubated (twice) at room temperature in NaCl solution (1M, 5 mL) for 15 min each, in methanol (5 mL), then in methanol-chloroform (1:1, 5 mL) twice for 30 min, and finally washed with methanol (5 mL). After each incubation, the supernatant was discarded.
The residue was dried in the open air and submitted to alkaline hydrolysis for 18 h at room temperature and in absence of light using NaOH (1M, 50 mL g-1). The alkaline extract was filtered and acidified with HCl until pH 3 and filtered again. The extract was submitted to the same process of cleanup with SPE described above, but the column was washed with ultrapure water and methanol 5% before elution using 10 mL of methanol.
2023-11-25 | MSV000093498 | GNPS
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