GNPS Metabolite diversity in two yacon cultivars
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Smallanthus Sonchifolius (ncbitaxon:185202)
SUBMITTER: 
Federico Padilla
PROVIDER: MSV000085101 | GNPS | Sat Mar 14 08:35:00 GMT 2020
REPOSITORIES: GNPS
Similar Datasets
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
Project description:Human primary macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 6 hrs or left untreated. Cells were collected and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the supernatant was collected and the protein content was measured with Bio-Rad DC™ protein assay (Bio-Rad). The proteins were reduced, alkylated, and enzymatically digested in-solution with trypsin. The digestion was stopped by adding FA (final c = 1 %). The samples were centrifuged 9168 x g for 10 min and desalted with Sep-Pak Vac RP C18 cartridges (Waters, MA, USA), following fractionation by strong cation exchange chromatography (SCX). The peptides were separated on a 200 x 4.6 mm, 5 μm, 200 Å PolySULFOETHYL A™ column (PolyLC, USA). The fractions were vacuum centrifuged and desalted as before, following phosphopeptide-enrichment with PHOS-Select™ Iron Affinity Gel (Sigma Aldrich, MO, USA). LC-MS/MS was performed with a Q Exactive hybrid quadrupole-orbitrap tandem mass spectrometer coupled to an EASY-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher Scientific). A 100 μm x 3 cm trap column and a 75 μm x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 μm; Michrom Bioresources). The mobile phases were 2% acetonitrile, 0.2% formic acid (A) and 95% acetonitrile, 0.2% formic acid (B). LC gradient elution condition was 2% B (0 min), 20% B (70 min), 40% B (100 min), and then 100% B (105-110 min), with a flow rate of 300 nl/min. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 300 to m/z 2000 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 120 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation (HCD) with an isolation window of 2 and normalized collision energy of 30. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 250 ms, and the lowest mass fixed at m/z 100. Dynamic exclusion duration was 30 s.
2016-08-08 | PXD001620 | Pride
Project description:Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Several previous transcript-profiling experiments have compared separate anaerobic and aerobic cultures. Here, for the first time, the process of adaptation was investigated by determining changes in transcript profiles when anaerobic steady-state cultures were perturbed by the introduction of air. Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 400 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (30 mM) as the carbon source with the dilution rate being 0.2 h-1. Anaerobic cultures were maintained by sparging the chemostat with oxygen-free nitrogen (95 %) and carbon dioxide (5 %) (0.2 l min-1). The switch to aerobic conditions was achieved by sparging the culture with air (2.0 l min-1) in place of the above gas mix. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions. Keywords: time course response to air
2006-07-28 | GSE4735 | GEO
Project description:Proteome quantification using TMT reagents and off-line basic peptide fractionation was done as described previously in Grand, 2021, with the exception that the isolated nuclei were used as a starting material rather than whole cells to enable detection of transcription factors which are generally less abundant proteins in the cell, and that nine channels from a TMTpro16 reagent kit were used. Briefly, mESCs, NGN2 24h induced cells and MyoD1 24h induced cells were harvested in triplicates of 10 million per sample. Nuclei were isolated by suspending in 4 ml of nuclear extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 10 mM EDTA, 0.5% NP-40, 1 mM DTT and complete protease inhibitor (Roche)) and incubating on ice for 10 min with vortexing every 2 minutes. Nuclei were collected by centrifugation (800g, 10 min, 4 °C), washed with ice cold PBS and suspended in 1% sodium deoxycholate in 50 mM HEPES buffer (pH 8.5). The nuclear lysate was disrupted with sonication. Proteins were reduced, alkylated and Lys-C/trypsin digested. Fifty micrograms of peptides from each biological replicate were labelled using channels 128C-131C from a TMT 16plex isobaric labelling reagents kit (Lot# VJ313476, Thermo Fisher Scientific). Dried TMT-labeled peptides were dissolved in 20 μL of 10 mM ammonium formate (pH 10, high pH Buffer A), and high-pH reversed-phase chromatography was employed using an Agilent 1100 HPLC system with an autosampler, a YMC Triart C18 0.5 x 250 mm column, and a fraction collector. The peptides were separated using the following binary buffer system: high-pH Buffer A (20 mM ammonium formate in water, pH 10) and high-pH Buffer B (20 mM ammonium formate pH 10 in 90% acetonitrile) at a flow rate of 12 μL/minute. The gradient duration was a total of 115 min, with mobile phase compositions in %B as follows: 0-5 min at 2%-15%, 5-15 min at 15%-25%, 15-80 min at 25%-45%, 80-85 min at 45%-65%, 85-95 min at 65%-100%, 95-100 min at 100%, 100-101 min at 100%-2%, and 101-115 min at 2%, resulting in 48 fractions after sample concatenation. Samples were acidified with 1% TFA and dried in a speed vac. Peptides reconstituted in 2% acetonitrile, 0.1% formic acid were on-line separated on a C18 50 cm μPAC column using a EASYnLC-1000 system (all Thermo Fisher Scientific) mounted on a Digital PicoView nanospray source (New Objective), connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptide elution was achieved using a linear gradient of increasing concentration of acetonitrile in 0.1% formic acid at a flow rate of 500 nl/min: 0-2 min: 2%, 2-8 min: 2-6%, 8-89 min 6-22%, 89-107 min: 22-40%, 107-111 min: 40-80%, 111-115 min: 80% (washing), 115-116 min: 80-2%, 116-120 min: 2% (equilibration). For MS data acquisition in a data-dependent mode, ions for survey MS1 scans were recorded in profile mode in the Orbitrap detector at a resolution of 120,000 in the range of 400-1400 m/z every 3 seconds for a maximum of 100 ms to reach the normalized AGC target of 50%. The most abundant precursors were selected in the quadrupole within an isolation window of 0.7 m/z for MS2 HCD fragmentation based on advanced peak determination, monoisotopic peak determination set to peptides, within charge states 2-8, dynamic exclusion of 60 s within +/-10 ppm tolerance for isotopes and different charge states, with a minimum intensity of 5e4, with normalized collision energy 34% for a maximum injection time of 200 ms to reach the normalized AGC target of 80%. MS2 HCD spectra were and recorded in the “normal” range, starting at 100 m/z at 50,000 resolution in the Orbitrap analyzer in centroid mode.
2025-08-21 | PXD054143 | Pride
Project description:The CLIP procedures were conducted using HeLa cells according to the previous study (Liu et al., 2016) with slight modifications. Briefly, 4 plates of HeLa cells in 15 cm dishes were transfected with pPB/ALKBH3 plasmid for 48 h to reach about 90% cell confluency. After washing twice with ice-cold PBS, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice. Cells were lysed in high salt lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor cocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4°C for 30 min. Supernatant was collected after centrifuged at 17,000 g at 4°C for 15 min and further treated with 1 U RNase T1 for 15 min at 24 °C. After centrifugation and filtration through 0.22 µM filter, 10% supernatant was collected as input for further sequencing analysis. Anti-Flag M2 beads (Sigma-Aldrich, St. Louis, MO, 80 μl) were washed three times with lysis buffer and incubated with the filtered supernatant at 4°C for 4 hrs. After removing the flow through by magnetic rack, beads and input were further incubated with 10 U RNase T1 exactly for 8 min. Samples were treated with 95 µL commercial PNK buffer and 0.5 U PNK for 15 min at 37 °C. After treatments, final concentration of 100 µM ATP and 1 U PNK were added and incubated at 37 °C for another 30 min. The RNA/protein complexes were eluted by NuPAGE 1 × loading buffer and fractionated by neutral NuPAGE 4–12% bis-tris gel. After cutting the gel, RNAs were recovered by proteinase K digestion in proteinase K buffer, followed by phenol/chloroform extraction using glycogen as carrier. The RNA fragments were ligated to adapter and established library by NEB small RNA library preparation kit (E7330S).
2022-03-01 | GSE112625 | GEO
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Briefly rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031).
2019-10-15 | GSE134103 | GEO
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
Project description:This study looked for early response signals produced in UV-B irradiated leaves and induced in shielded leaves and shielded ears that modulate physiological responses in maize. Transcriptome profiling tracked changes in exposed and shielded organs. Metabolic profiling was examined for signaling molecules. The top 2 leaves were exposed to 10, 30, or 60 minutes of ~3x normal UV-B and then tissue samples were taken from all 3 tissue types. Over 250 transcripts are differentially expressed at least 2 fold in irradiated leaves by 10 min of UV-B. At 30 min this decreased to ~150 then increased to over 500 transcripts at 60 min. Different pathways are affected at the various time exposures and specific responses are modulated by the time of exposure. Shielded leaves had a smaller number of transcripts affected at each time point but over 60% were in common with the irradiated leaves results at 10 min. and ~20% overlaps were found at 30 and 60 min. Metabolite samples were analyzed after 5, 10, 15, 30, and 90 min of UV-B. We identified 14 compounds with a statistically significant change from untreated plants in at least one time point.
2011-07-31 | GSE30278 | GEO
Project description:Leaf and stem plant material (0.2 g fresh weight) was collected from aerial tissues, frozen and ground with a MP Biomedicals FastPrep-24-5G Tissuelyser in 2ml MP Biomedicals tubes with 2.3mm Zirconia beads for 60 s at 6 m/s. 1 mL of 80% methanol was added to ground tissue and ground as before for 20 s at 6 m/s. The methanolic extract was incubated for 10 min in a 60C water bath, centrifuged for 5 min at 16000 x g at room temperature, and filtered through Whatman 0.2 um syringeless LCMS filters (UN503NPEORG) before LCMS analysis. Filtered extracts were transferred to LCMS vials and analyzed by LC-MS/MS with the following settings: LC - Phenomenex Kinetex 2.6 um C18 reverse phase 100A 150 x 3 mm LC column; LC gradient, solvent A, 0.1% formic acid; solvent B, acetonitrile (0.1% formic acid); 0 min, 10% B; 5 min, 60% B; 5.1 min, 95% B; 6 min, 95% B; 6.1 min, 10% B; 9.9 min, 10% B; 0.5 mL/min; MS, positive ion mode; full MS, resolution 70000; mass range 400-1200 m/z; dd-MS2 (data-dependent MS/MS), resolution 17500; AGC target 1e5, loop count 5, isolation width 1.0 m/z, collision energy 25 eV and dynamic exclusion 0.5 s.
2024-10-14 | MSV000096083 | MassIVE
Project description:To probe the binding sites of MeCbl on GSDME, 0.4 μM recombinant human GSDME (CSB-EP006766HU) purchased from CUSABIO (Wuhan, China) was incubated with 10 μM MeCbl in 100 μL buffer (20 mM HEPES, 150 mM NaCl and 50 μM DPA) at room temperature for 2 h and protected from light. 0.4 μM recombinant human GSDME was incubated with ddH2O as negative control, or with HOCbl for binding sites analysis between GSDME and cobalamin. All groups were equally prepared ten incubation samples and mixed for further analysis. Trypsin and Glu C were then added in the mixture overnight, and formic acid was added to adjust the pH down to three to terminate the enzyme digestion. Activated the C18 desalting column with 100 μL 100% acetonitrile, then centrifuged at 400 g for 3 min. Added 100 μL of 0.1% formic acid and centrifuged at 400 g for 3 min for column equilibration. Replaced with new EP tube, added samples to column, and centrifuged at 400 g for 3 min. Washed the column twice with 100 μL of 0.1% formic acid and centrifuged at 400 g for 3 min, washed once again with 100 μL water at pH10. The eluents were collected with 70% acetonitrile, each sample was lyophilized and stored at -80°C until further mass spectrometry analysis. Prepared the mobile phase solution, A (0.1% (v/v) formic acid in ddH2O), B (0.1% (v/v) formic acid and 80% acetonitrile in ddH2O), the lyophilized samples were dissolved with 10 µL solution A, then centrifuged at 14000 g for 20 min at 4°C, 400 ng supernatant of the samples were injected onto a Thermo Scientific™ UltiMate™ 3000 UHPLC coupled to a timsTOF HT mass spectrometer (BRUKER). Peptides were then separated over a 100 μm ⅹ15 cm ReproSil-Pur C18-AQ 1.5-µm silica beads (Beijing Qinglian Biotech Co.,Ltd, Beijing, China), at a flow rate of 300 nL/min using a gradient: 0-4 min (5-10% B), 4-46 min (10-24% B), 46-53 min (36% B), 53-54 min (95% B), 54-60 (95% B). The timsTOF HT mass spectrometer was used for liquid quality detection with Captive Spray source, and the mass spectrum was collected in DDA mode. The scanning range of the mass spectrum was m/z 100-1700, the primary mass spectrum resolution was set to 60,000 (1222 m/z), and the cumulative time was set to 100 ms in the TIMS tunnel. The capillary voltage is set at 1.6 kV and the mobility is 0.6-1.6 cm2/(V). The total cycle time was 1.1s with 10 PASEF cycles. The mass spectrometry raw data was processed by FragPipe to search against the target protein database. The precursor ion mass tolerance was ±15 ppm; the fragment mass tolerance was ±0.02 Da. The static modification was carbamidomethylation of cysteine; the dynamic modifications were oxidation of methionine, acetylation of N-termini of peptides and C63H91CoN13O14P or C62H88CoN13O14P of cysteine; a maximum of two missed cleavages were allowed.
2024-11-11 | PXD057768 |