Dataset: Intracellular metabolites
Deployment: KN210-04

Metadata from "MTBLS461: Intracellular metabolites from an experimental manipulation of marine microorganisms"

at https://www.ebi.ac.uk/metabolights/MTBLS461/samples

Sample Collection:
 
Seawater for the incubation experiments was collected using 10 l Niskin bottles attached to a CTD/rosette system. Seawater was collected off the northeastern corner of South America at 9.75 N, 55.3 W from 70 m on May 5th, 2013. Silicone tubing was used to collect water from the Niskins and the tubing was placed in the bottom of polycarbonate carboys in order to minimize turbulence during sample collection. The seawater was first filtered through a 0.2 µm Sterivex (Millipore) to obtain cell-free seawater. To obtain cell- and virus-free seawater, tangential flow filtration using a recirculating Prep/Scale tangential flow ultrafilter (Millipore) with a 30 kDa molecular mass cut-off was used.

5 different experimental treatments were established: 1) a whole seawater control, 2) 20% whole seawater with cell-free seawater, 3) 45% whole seawater with cell-free seawater, 4) 20% whole seawater with cell- and virus-free seawater, 5) 45% whole seawater with cell- and virus-free seawater. There were 3 x 2 l polycarbonate bottles established for each treatment. Each bottle held 2320 ml of fluid. 1 of the bottles was sampled immediately after the experiment was set up. The 2 remaining bottles were incubated for 1 day in an on-deck, flow-through incubator that allowed 10% of photosynthetically active radiation (PAR) to pass through its screening.

Extraction: 

The intracellular metabolites were extracted using a method modified from a previously desbribed protocol (Rabinowitz & Kimball, 2007) . Briefly, the filter was extracted 3 times with ice-cold extraction solvent (acetonitrile:methanol:water with 0.1 M formic acid, 40:40:20). The combined extracts were neutralized with ammonium hydroxide and dried in a vacufuge.
The samples for the targeted mass spectrometry analysis were re-dissolved in 95:5 (v/v) water:acetonitrile and combined with deuterated biotin (final concentration 0.05 µg/ml) as an internal standard. The final extract volume was 100 µl.

For untargeted analysis, the extracts had to undergo an additional de-salting step prior to analysis. Therefore, the dried extracts were re-dissolved in 0.01 M hydrochloric acid and extracted using a 50 mg/1 cc PPL cartridge following a previously established protocol (Dittmar et al., 2008). The resulting methanol extracts were re-dissolved in 95:5 water:acetonitrile and deuterated biotin.
Chromatography:

For targeted metabolomics analysis, the samples were analyzed with a Synergi 4µ Fusion – RP 80A 150 x 2.00 mm column (Phenomenex, Torrance, CA) coupled to a Thermo Scientific TSQ Vantage Triple Stage Quadrupole Mass Spectrometer. The chromatography gradient was: an initial hold of 95% A (0.1% formic acid in water):5% B (0.1% formic acid in acetonitrile) for 2 min, ramp to 65% B from 2 to 20 min, ramp to 100% B from 20 to 25 min, and hold until 32.5 min. The column was re-equilibrated for 7 min between samples with solvent A. Each metabolite was quantified using multiple reaction monitoring (MRM) mode with optimal parameters determined from infusion of authentic standards. 10-point external calibration curves (0.5, 1, 5, 10, 25, 50, 100, 250, 500, and 1000 ng/ml) were generated for each compound by plotting peak area against concentration.
For untargeted metabolomics analysis, LC separation was performed using a Synergi Fusion reversed phase column (Phenomenex, Torrance, CA) with the same gradient as for targeted analysis.  

Mass spectroscopy:

For targeted analysis, samples were analyzed with a Thermo Scientific TSQ Vantage Triple Stage Quadrupole Mass Spectrometer. This instrument allows polarity switching between positive and negative ion mode within a single LC run. Analysis of authentic standards was used to determine the optimal ionization mode for each metabolite.

For untargeted analysis, samples were analyzed in negative ion mode with liquid chromatography (LC) coupled by electrospray ionization to a 7-Tesla Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR MS). In parallel to the FT acquisition, 4 data dependent MS/MS scans were collected at nominal mass resolution in the ion trap (LTQ). Samples were analyzed in random order with a pooled sampled run every 6 samples in order to assess instrument variability.

Metabolite identification:

The targeted metabolomics compound identifications were based on measurements of authentic standards on the same mass spectrometer. All identifications were 'MSI Level 1' identifications based on the established criteria (Sumner et al., 2007).