The procedure is divided in three sections (I. Cultivation / Sampling; II. Sample preparation for the determination of cellular DMSOP; III. Chromatography and mass spectrometry). Details for each section are described below. Note: The protocol generates samples that allow the determination of other low molecular weight zwitterionic metabolites like DMSP, glycine betaine or gonyol. For these and other zwitterionic metabolites the protocol can be followed with only one adjustment: appropriate standards of the analytes of interest would have to be used instead of the DMSOP standard.
I. Cultivation / Sampling
Utilize any culture of microalgae or field sample of phytoplankton. The amount of culture / field sample depends on the cell count, cell volume, and the content of DMSOP or of other zwitterionic low molecular weight metabolites of the tested alga. Further, the sensitivity of the LC/MS equipment has to be taken into consideration. The protocol introduced below has been optimized for 100 – 200 mL exponential phase cultures or 20 – 50 mL stationary phase cultures of diatoms (here Skeletonema costatum) or 100 µL of dense bacterial cultures (here Pelagibacter bermudensis) and measurement on an Orbitrap Q-Exactive LC/MS instrument. The protocol can be readily scaled up and down.
II. Sample preparation for the determination of cellular DMSOP
Make sure you have ready the equipment and solutions before the start of the procedure. Label the vials before the start of the experiment. The workflow should be carried out without interruption if quantitative information is required. If required, the protocol could only be paused after point 5. (A) or 3. (B).
A) Extraction of phytoplankton cells
- Take a specific volume from your experimental culture using a serological pipette and transfer it into an Erlenmeyer flask.
Note: The volume is dependent on growth phase and cell density: for exponential phase diatom cultures use 100 – 200 mL, for stationary phase 20 – 50 mL.
Note: Determine cell counts and / or chlorophyll a content of the culture and document the precise volume. This information will be required to calculate the cellular DMSOP content / the DMSOP concentration.
- Filter the culture under reduced pressure (400 mbar) over a GF/C grade microfiber filter using the filtration unit. Use a two-way stopcock to control the vacuum. Close the stopcock as soon as the filter is dry.
Note: The filtration should be carried out quickly and the filter should be transferred directly once the filtration is terminated.
- Remove the glass part of the filtration unit and fold the filter on the glass frit two times using tweezers.
- Using tweezers transfer the filter to a 4 mL screw top glass vial containing 1 mL of methanol.
- Seal the vial with a Teflon lined screw cap and vortex it for 30 s.
Note: You can store the sample at 20 °C for several weeks without loss (for long-term storage over several months’ 80 °C storage might be recommended). Note: If you notice precipitate formation after freezing centrifuge the sample and transfer the supernatant as described in step 6.
- Transfer 50 µL of the extract into a micro glass insert and dilute with 100 µL of a mixture of acetonitrile and water (9:1 v/v).
Note: The dilution can be adjusted depending on the concentration of the extract.
Note: If you use an internal standard (procedure III B), add it here.
- Put the micro glass insert into a 15 mL Falcon tube and centrifuge for 5 min at 4500 rcf. Transfer the supernatant to another micro glass insert.
Put the insert with the probe into a 1.5 mL screw top glass vial equipped with a spring to adjust the insert, close the lid and put it into the autosampler of the LC/MS system. Inject 5 µL to the LC/MS.
B) Extraction of bacterial cells
- Take 100 µL of the bacterial culture and transfer it to a 1.5 mL Eppendorf tube.
- Centrifuge it for 5 min at 16,100 rcf.
Note: If you do not see a cell pellet repeat the centrifugation for another 30 min.
- Remove and discard the supernatant by pipetting.
Pipette 100 µL of a mixture of acetonitrile and water (9:1 v/v) on the cell pellet and vortex for 30 s.
Note: If you use an internal standard (procedure III B), add it here.
Note: You can store the sample at -20 °C for several days (for long-term storage over several months’ 80 °C storage might be recommended).
- Put the tube on ice and disrupt the cells by sonication using six 10 s pulses (40% intensity) in the ultrasound homogenizer.
- Centrifuge the sample for 5 min at 16,100 rcf and transfer the supernatant into the micro glass insert of a 1.5 mL screw top glass vial equipped with a spring to hold the insert and close the lid. Note: The supernatant has to be free of any precipitate; re-centrifuge if required.
Inject 5 µL into the LC/MS system.
III. Chromatography and Mass spectrometry
Chromatography
• Solvent A: high purity water with 2% acetonitrile and 0.1% formic acid.
• Solvent B: 90% acetonitrile with 10% water and 5 mmol L-1 ammonium acetate.
• Column: SeQuant ZIC®-HILIC column (5 μm, 2.1 × 150 mm, SeQuant, Umeå, Sweden), SeQuant ZIC®-HILIC guard column (5 μm, 2.1 × 20 mm, SeQuant, Umeå, Sweden).
• Column oven temperature: 25 °C.
• Flow rate: 0.6 mL min-1
• Gradient: linear, 100% solvent B (1 min), 20% B (6.5 min), 100% B (7.1 min), 100% B (10 min)
• Injection volume: 5 µL
Mass spectrometry
• Ionization: electrospray ionization, positive mode
• Mass range: 75 – 200 m/z
Exactive™ Plus Orbitrap mass spectrometer (high resolution MS):
• Capillary temperature: 380 °C
• Spray voltage: 3000 V
• Sheath gas flow: 60 arbitrary units
• Aux gas flow: 20 arbitrary units
Q-ToF micro mass spectrometer:
• Capillary temperature: 300 °C
• Spray voltage: 3000 V
• Sample cone voltage: 18 V
• Extraction cone voltage: 1 V
• Sheath gas: 20 L h-1
• Desolvation gas: 450 L h-1
• Detector voltage: 2350 V
A) Quantification using external standard
For external calibration of DMSOP prepare a concentrated stock solution of 10 µmol L-1 in acetonitrile and water (9:1 v/v). Perform dilutions to obtain 1000 nmol L-1, 500 nmol L-1, 300 nmol L-1, 50 nmol L-1, 10 nmol L-1, 5 nmol L-1, 1 nmol L-1, 0.5 nmol L-1 DMSOP solutions. Each concentration is independently prepared in triplicate in acetonitrile and water (9:1 v/v). Note: For quantification of other zwitterions prepare similar dilution rows with the respective standard (e.g. DMSP, gonyol, DMSA, glycine betaine6)
Dependent on the DMSOP content in the respective samples you can vary the concentration range of the calibration curve. Injection volume for LC/MS measurements is 5 µL.
Monitoring the ion trace of the respective analyte and integration of the peak gives the peak area.
DMSOP m/z = 151.0496 (isolation window 5 ppm) for high resolution MS or m/z = 151 for unit mass resolution MS
DMSP m/z = 135.0474 for high resolution MS or m/z = 135 for unit mass resolution MS.
Gonyol m/z = 179.0736 for high resolution MS or m/z = 179 for unit mass resolution MS.
DMSA m/z = 121.0318 for high resolution MS or m/z = 121 for unit mass resolution MS.
Glycine betaine m/z = 118.0863 for high resolution MS or m/z = 118 for unit mass resolution MS.
B) Quantification using internal standard
For internal calibration of DMSOP prepare a concentrated stock solution of the internal standard 2[H]6-DMSA (16 µmol L-1) in acetonitrile and water (9:1 v/v).
Add 10 µL of that solution to your prepared sample (see Procedure II A, step 6) and directly inject 5 µL to your LC/MS system.
Note: The ratio of internal standard and signal of the respective analytes should be similar. If differences >10/1 are observed the concentration has to be adjusted by using less standard. If differences are <1/10 the sample has to be diluted.
Measuring a DMSOP and 2[H]6-DMSA containing solutions gives the response factor (ADMSOP/A2[H]6--DMSA) you need for quantification of DMSOP. Note: Make sure you are working in the linear range of the detector.
For quantification of DMSP use 2[H]6-DMSP as internal standard6, for DMSA 2[H]6-DMSA and for gonyol and glycine betaine use D3-gonyol6.
Maintenance of the LC-MS system
• Instrument tuning: every 3 months
• Mass calibration: weekly
• Wash solutions (syringe etc.): change every month
• Ion source cleaning: weekly
• Worn out parts (injection needle, column, seals etc.): replace as needed