Dataset Information

Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean

ABSTRACT: The 15 strains of Gambierdiscus were cultivated in four separate laboratories. Each laboratory grew their available strains (see sample metadata) in addition to the G. australes strain AUS S080911_1 isolated from Pacific Ocean. Irradiance (70-100 umol photons m-2 s-1) and light/dark cycle (12h:12h) were standardized between the different laboratories, the other cultivation parameters were defined by each laboratory considering optimal growth parameters and technical requirements. Samples were extracted twice in methanol 90% from freeze-dried cell pellets (2mL per million of cells). The extraction cycle was as follows: vortex 30 s, ultrasonic bath (25 kHz on ice, 15 min), vortex 30 s and centrifugation (4 000 g, 2 min). The extracts resulting from the two extraction cycles were pooled into an amber glass vial and stored at -80 C (final concentration : 250,000 cells mL-1). Metabolomic profiles were acquired by Ultra-high-performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS). The instrumentation consisted of a UHPLC system (1290 Infinity II, Agilent) coupled to a quadrupole-time of flight mass spectrometer (QTOF 6550, Agilent) equipped with a Dual Jet Stream electrospray ionization (ESI) interface. The analytical column was a core-shell Kinetex C18 (100 x 2.1 mm, 1.7 um, Phenomenex) with a suited guard column. Mobile phases consisted of water (A) and acetonitrile/water (95:5, V:V) (B), both containing 2 mM ammonium formate and 50 mM formic acid The flow rate was 0.4 mL min-1 and the injection volume was 5 uL. The following elution gradient was used: 5% B (0-1 min), 5-100% B (1-11 min), 100% B (11-13 min), 5% B (13-18 min). Mass spectra were recorded in both positive and negative full-scan modes from m/z 100 to 1700 at a mass resolving power of 25 000 full-width at half-maximum (fwhm, m/z = 922.0099) and an acquisition rate of 2 spectra/s. Auto-MS/MS was performed iteratively: each sample was injected 5 times in ESI+ mode and the ions fragmented were manually excluded from the following analyses

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Gambierdiscus Silvae (ncbitaxon:1550089) Gambierdiscus Carolinianus (ncbitaxon:864182) Gambierdiscus Australes (ncbitaxon:439317) Gambierdiscus Belizeanus (ncbitaxon:439316) Gambierdiscus Excentricus (ncbitaxon:986170) Gambierdiscus Caribaeus (ncbitaxon:864185)

SUBMITTER: Thomas Yon

PROVIDER: MSV000094172 | MassIVE | Mon Feb 26 10:31:00 GMT 2024

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Publications

Targeted and non-targeted mass spectrometry to explore the chemical diversity of the genus Gambierdiscus in the Atlantic Ocean.

Yon Thomas T Réveillon Damien D Sibat Manoëlla M Holland Chris C Litaker R Wayne RW Nascimento Silvia M SM Rossignoli Araceli E AE Riobó Pilar P Hess Philipp P Bertrand Samuel S

Phytochemistry 20240416

Dinoflagellates of the genus Gambierdiscus have been associated with ciguatera, the most common non-bacterial fish-related intoxication in the world. Many studies report the presence of potentially toxic Gambierdiscus species along the Atlantic coasts including G. australes, G. silvae and G. excentricus. Estimates of their toxicity, as determined by bio-assays, vary substantially, both between species and strains of the same species. Therefore, there is a need for additional knowledge on the met ...[more]

PMID: 38631521

Similar Datasets

Project description:The 15 strains of Gambierdiscus were cultivated in four separate laboratories. Each laboratory grew their available strains (see sample metadata) in addition to the G. australes strain AUS S080911_1 isolated from Pacific Ocean. Irradiance (70-100 umol photons m-2 s-1) and light/dark cycle (12h:12h) were standardized between the different laboratories, the other cultivation parameters were defined by each laboratory considering optimal growth parameters and technical requirements. Samples were extracted twice in methanol 90% from freeze-dried cell pellets (2mL per million of cells). The extraction cycle was as follows: vortex 30 s, ultrasonic bath (25 kHz on ice, 15 min), vortex 30 s and centrifugation (4 000 g, 2 min). The extracts resulting from the two extraction cycles were pooled into an amber glass vial and stored at -80 C (final concentration : 250,000 cells mL-1). Metabolomic profiles were acquired by Ultra-high-performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS). The instrumentation consisted of a UHPLC system (1290 Infinity II, Agilent) coupled to a quadrupole-time of flight mass spectrometer (QTOF 6550, Agilent) equipped with a Dual Jet Stream electrospray ionization (ESI) interface. The analytical column was a core-shell Kinetex C18 (100 x 2.1 mm, 1.7 um, Phenomenex) with a suited guard column. Mobile phases consisted of water (A) and acetonitrile/water (95:5, V:V) (B), both containing 2 mM ammonium formate and 50 mM formic acid The flow rate was 0.4 mL min-1 and the injection volume was 5 uL. The following elution gradient was used: 5% B (0-1 min), 5-100% B (1-11 min), 100% B (11-13 min), 5% B (13-18 min). Mass spectra were recorded in both positive and negative full-scan modes from m/z 100 to 1700 at a mass resolving power of 25 000 full-width at half-maximum (fwhm, m/z = 922.0099) and an acquisition rate of 2 spectra/s. Auto-MS/MS was performed iteratively: each sample was injected 5 times in ESI+ mode and the ions fragmented were manually excluded from the following analyses

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.

Project description:1ml of WT, delta-pka, delta-arcA and delta-pka delta-arcA culture broth was harvested at u=0.4 h-1, centrifuged (14000 x g for 1 min at 4 degrees C), pellet washed once with PBS, flash frozen with liquid N2 and kept at -80 degrees C until further processing. Frozen pellets were melted on ice after which 100 ug of sample biomass was mixed with 100 ug of SILAC-labeled E. coli biomass (internal standard) and frozen at -80 degrees C. Cell pellets were suspended in 100 uL SDS lysis buffer (4% SDS/100 mM TRIS-HCl pH 8/100 mM DTT) and heated at 95 degrees C for 15 min. Cell lysates were sonicated with ultrasound for a few pulses and pelleted by centrifugation. Cell lysates were digested with trypsin according to Filter Aided Sample Preparation protocol (FASP) (Wiśniewski et al. 2009) and purified with C-18 StageTips (Rappsilber et al. 2007). LC-MS/MS analysis was performed using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer (Thermo Electron, San Jose, CA, USA) equipped with a nanoelectrospray ion source (Proxeon, Odense, Denmark). Peptides were separated with a 240-minute gradient from 2 to 40% B (A: 0.5% acetic acid, B: 0.5% acetic acid/80% acetonitrile) using a flow-rate of 200 nl/min. Data analysis of raw MS-files was performed by the MaxQuant software package version 1.3.0.5 (Cox and Mann 2008). Peak lists were searched using the Andromeda search engine (built into MaxQuant) against an E. coli database (downloaded 09.04.2013 from http://www.uniprot.org/) which was supplemented with common contaminants (e.g human keratin, trypsin). Full tryptic specificity, a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ions was specified in the MaxQuant search. Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation and protein N-terminal acetylation were set as variable modifications. The required false discovery rate was set to 1% for both peptide and protein levels and the minimum required peptide length was set to six amino acids. “Match between runs” option with a time window of 1.5 min was allowed.

Project description:Experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Microarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice. Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm). The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C. Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C. After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm. Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms. Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments. The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software. Keywords: other