In vivo DMSP‐biosynthesis measurements using stable‐isotope incorporation and proton‐transfer‐reaction mass spectrometry (PTR‐MS)

Abstract

Assessing turnover rates of dimethylsulfoniopropionate (DMSP), precursor of the climate-active gas dimethylsulfide (DMS), under different environmental conditions is fundamental to accurately modeling the marine sulfur cycle. Current gas chromatographic methods only provide net results of all processes acting on the different sulfur pools and are unable to measure fluxes, leaving room for speculation on the controlling factors and magnitudes of turnover processes. Here we present a new method in which stable isotope additions (D 2 O or NaH 13 CO 3 ) are used to follow the in vivo production of DMSP. Incorporation of deuterium or 13 C into DMSP was monitored using a proton-reaction-transfer mass spectrometer (PTR-MS). A growth model was developed to calculate specific DMSP synthesis rates from the progress in mass ratio of labeled versus nonlabeled DMSP over time. Instead of measuring the amount of incorporated isotope, our method uses ratios of molecule masses. This approach has the advantage that the effect of the isotope addition on the change in mass ratio is amplified by the number of positions the label can occupy within the molecule. Application of the method was demonstrated in cultures of the ubiquitous DMSP producer Emiliania huxleyi and further developed for use in short-time incubation studies with water from the Sargasso Sea during July 2004. Very low specific synthesis rates between 0.05 and 0.2 d -1 could be measured. The method is the first to measure in vivo DMSP production rates. It is relatively easy to apply, highly sensitive, and may also be applicable to other biogenic compounds.

Contrasting responses of DMS and DMSP to ocean acidification in Arctic waters

“…Determination of de novo DMSP synthesis rates (µDMSP) was carried out according to Stefels et al (2009). Incorporation of 13 C into DMSP was determined by proton transfer reaction mass spectrometry (PTR-MS) of DMS swept from the 20 mL vials and recorded as mass 63, 64 and 65 of protonated forms of 12 C-DMS, 13 C-DMS 34 S-DMS, respectively.…”

“…A weighted average approach that gives most weight to the initial points of the exponentially decreasing DMS peak was used to calculate the mass ratio 64 MP t for each sample at each time point. The mass ratio progress method described by Stefels et al (2009) was applied to calculate µDMSP. This applied information from culture-based studies of Emiliania huxleyi to calculate the isotope fractionation factor (Stefels et al, 2009).…”

“…The mass ratio progress method described by Stefels et al (2009) was applied to calculate µDMSP. This applied information from culture-based studies of Emiliania huxleyi to calculate the isotope fractionation factor (Stefels et al, 2009). The exact tracer addition was calculated from the weight of NaH 13 CO 3 added and the daily measurements of DIC in the mesocosms.…”

Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria

“…This procedure, applicable to any system, may serve as a template to study the sub-cellular localization and identification of other small and highly diffusible molecules. In addition, similarly to other recent stable isotope approaches (Stefels et al, 2009), our method may be used to quantify the production rate of DMSP at the single cell level. We confirmed that 34 S-DMSP was the main organic sulfur compound within the algal cells and we subsequently localised large quantities of the sulfur tracer 34 S in algal vacuole, cytoplasm and chloroplasts.…”

Combining cell sorting with gas chromatography to determine phytoplankton group-specific intracellular dimethylsulphoniopropionate

“…These include X-ray microanalysis (Heldal et al 1985), modified isotope ratio mass spectrometry (IRMS) (Eek et al 2007) and secondary ion mass spectrometry (SIMS) (Orphan & House 2009). The latter 2 approaches potentially provide estimates of stable isotopic composition of small sample sizes, opening up the potential to combine FACS-GC based estimates of intracellular DMSP content in specific groups with measures of their metabolic activity, including possibly in vitro DMSP synthesis (Stefels et al 2009). One advantage of the mechanical-FACS mechanism is the capability to use radioisotope tracers to determine metabolic activity, including DMSP uptake, in specific components of microbial populations , Vila-Costa et al 2006 without the problems associated with production of radioactive aerosols that droplet-FACS mechanisms potentially produce.…”