Metabolomic study of saxitoxin analogues and biosynthetic intermediates in dinoflagellates using 15N-labelled sodium nitrate as a nitrogen source

A stable-isotope-labelling method using 15N-labelled sodium nitrate as a nitrogen source was developed for the toxic dinoflagellate Alexandrium catenella. The labelled saxitoxin analogues (STXs), their precursor, and the biosynthetic intermediates were analyzed by column-switching high-resolution hydrophilic interaction liquid chromatography with mass spectrometry. The low contents on Day 0, high 15N incorporation % of Int-C’2 and Int-E’ suggested that their turn-over rates are high and that the sizes of the pool of these compounds are smaller than those of the other intermediates. The experimentally determined isotopomer distributions showed that arginine, Int-C’2, 11-hydroxy-Int-C’2, Int-E’, GTX5, GTX4, C1, and C2, each existed as a combination of three populations that consisted of the non-labelled molecules and the labelled isotopomers representing molecules newly synthesized by incorporation of 15N assimilated from the medium with two different incorporation rates. The order of 15N incorporation % values of the labelled populations predicted by this model largely agreed with the proposed biosynthetic route. The stable-isotope-labelling method will be useful for understanding the complex mechanism of nitrogen flux in STX-producing dinoflagellates.

Relative % of peak area of each isotopomer of the precursor and the biosynthetic intermediates at 3, 6, and 10 days after the addition of 15 N-NO 3 medium to A. catenella S18 Figure S-17. Relative % of peak area of each isotopomer the biosynthetic intermediates, 11-hydroxyl-Int-C'2 and Int-E' at 3, 6, and 10 days after the addition of 15 N-NO 3 medium to A. catenella S19 Figure S-18. Relative % of peak of each isotopomer of the STXs at 3, 6, and 10 days after the addition of 15 N-NO 3 medium in A. catenella S20 Figure          Intens.

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Optimization of Chromabond R HILIC SPE conditions using the non-labelled standard The SPE treatment for sample preparation prior to the HR-HILICquadrupole time-of-flight (Q-Tof) MS was modified from the previously described method developed for STXs [43]. Since the ZIC-HILIC R SPE used in the original method is not commercially available so far, this reagent was replaced with Chromabond R HILIC, a sorbent that has the same functional group. Moreover, the recovery rate of the biosynthetic intermediates was very low by the original method [43], which therefore was modified to permit the simultaneous analysis of both the labelled biosynthetic intermediates and the STXs. In the previous paper [15], cell pellets were lyophilized to recover the relatively less-polar compounds corresponding to the biosynthetic intermediates from early stages of the pathway, namely arginine, Int-A', and Int-C'2. For example, recovery of Int-C'2 was 5% by the original procedure [15]. Moreover lyophilization is time-consuming and difficult to apply for the multiple samples expected from a time-course study. Therefore, a clean-up procedure without lyophilization was developed. Simply performing the original procedure without lyophilization yielded low amounts of Int-A' and Int-C'2 (Supplementary Information Table S-2, Entry 1). The use of THF for application and washing of the sample yielded improved recovery of these biosynthetic intermediates. However, attempts at elution with 0.5 M acetic acid or 0.2 M formic acid directly after THF washing did not permit recovery of GTX4, C1, or C2 (Entry 2). Stepwise washing with THF, acetonitrile, and 95% acetonitrile containing 0.1% formic acid improved the elution of these STXs with 0.2 M formic acid (Entry 3). The recovery rates of the main toxins and the biosynthetic intermediates from 50 mg of Chromabond R HILIC adsorbent were determined using a standard mixture prepared at a concentration range similar to that observed experimentally in the cell extracts of dinoflagellate cultures. The elution volume was set to 200 L to enable direct analysis without the need for a concentration step, although the recovery rate could be improved by using a higher volume of elution solution.

Optimized sample clean-up for HR HILIC-ESI-Q-tof-MS and MS/MS
Aliquots of the harvested cultures were used to obtain cell counts by microscopy. The cultures (20 mL each) then were centrifuged at 1,700 g for 5 min at 4°C to pellet the cells. After removal of the supernatant, the pellet was transferred to a new micro-tube, re-suspended, and pelleted again by centrifugation. After removal of the supernatant, the pellet was resuspended in 300 μL of 0.5 M acetic acid. Samples were stored at -30°C until use. After thawing on ice, the cell suspension was subjected to sonication (three cycles at 100 Hz, 40% amplitude, for 30 s on ice with 30 s intervals). The homogenate was centrifuged at 20,000 g for 5 min at 4°C. The supernatant of each sample was subjected to ultra-filtration (Ultra-Free C3LGC, 10,000-Da cut-off, Millipore) at 4°C. An aliquot (100 μL) of the resulting filtrate was transferred to a new tube and mixed with three volumes of THF. In parallel, a column of Chromabond R HILIC adsorbent (50 mg, MACHEREY-NAGEL) was generated by packing into a disposable empty cartridge (syringe type cartridge (CS0111, S size) and frits (CF0003), Tomoe, Amagasaki, Japan) and conditioned with 200 μL of MilliQ water and 1 mL of THF. The sample was loaded onto the column and the column was sequentially washed with 500 μL of THF, 500 μL of CH 3 CN, and 500 μL of CH 3 CN/water/HCOOH (95:5:0.1, v/v/v). The column was eluted with 200 μL of 0.2 M HCOOH and an aliquot of the eluate (10 or 20 μL) was subjected to LC-MS.
For the MS/MS sample, a Chromabond R HILIC polypropylene column (500-mg) was pre-conditioned with 1 mL of MilliQ water and 5 mL of THF. The total extract from a 20-mL culture was loaded onto the column, and the column then was sequentially washed with 3 mL of THF, 3 mL of CH 3 CN, and 3 mL of CH 3 CN/water/HCOOH (95:5:0.1, v/v/v). The column was eluted with 3 mL of 0.2 M HCOOH and the eluate was concentrated under a stream of nitrogen gas. After reconstitution with 100 μL of MilliQ water, the sample was filtered through a Cosmospin filter H (0.45 μm) and an aliquot of the eluate (10 μL) was subjected to LC-MS/MS .

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Validation by the highly labelled sample mixed with the nonlabelled standard The two-month exposure was initiated in the same manner as the timecourse study and passage was carried out three times for a total interval of two months (each passage was performed at 2 weeks). After 2 months, the cell cultures had achieved a cell density of 5 x 10 3 cells mL -1 and aliquots (60 mL each) were harvested by centrifugation at 890 g for 3 min at 4 C. The supernatants were decanted and discarded. Each cell pellet was resuspended in 300 L of 0.5 M acetic acid and stored at −30 C until analysis. The mono-isotopic ions of the non-labelled compounds were not detected except for arginine; the completely labelled isotopomers constituted the primary peak for each compound (Supplementary Information Fig. S-13). After ultrafiltration, the filtrate was mixed with a standard solution containing arginine, Int-A', Int-C'2, C1, C2, and GTX1-5 at final concentrations of 5.0, 0.5, 0.5, 4.9, 1.1, 7.6, 2.2, 0.8, 2.6, and 3.5 M, respectively. The same procedure was performed for the control (without standard) sample and the standard mixture only. The recovery rates were calculated as follows: the area of mono-isotopic ion in the fortified sample minus that of the control was divided by that of the standard solution treated the same as the fortified sample. The samples for the validation study were prepared in triplicate. For the evaluation of matrix effects, the eluates of un-mixed cell extract from Chromabond R HILIC sorbent were mixed with the standard solution. The matrix effects were calculated as follows: the area of mono-isotopic ion in the mixed sample minus the area of the control was divided by that of the standard solution. The values of recovery rate and matrix effect of Int-C'2 and those of arginine were used for Cyclic-C', 11-hydroxyl-Int-C'2 and Int-E'. The relative % was calculated as follows: the area of mono-isotopic ion in the mixed sample minus that of the control was divided by the sum of the areas of all isotopomers containing 15 N as observed in the EICs of the fortified samples.

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Calculation of peak area removing the contribution of the naturally occurring stable isotopes The concept of calculation Arginine, Int-A', 11-hydroxy-Int-C'2, Int-E' and consist of C, H, O, and N. Int-C'2 consists of C, H, and N. STXs (GTX4, GTX5, C1, and C2) consist of C, H, O, N, and S. Therefore, the 15 N-labelled compounds contain not only the incorporated 15 N, but also the naturally occurring stable isotopes such as 13 C, 2 H, 17 O, 18 O, 15 N, 33 S, and 34 S. To obtain the newly synthesized isotopomer peak areas, it is necessary to remove the contribution by these naturally occurring stable isotopes. Since the natural abundances of the stable isotopes 13 C, 15 N, 18 O, and 34 S are 1.07, 0.364, 0.205 and 4.25%, respectively, two different isotopomers with the same nominal mass can exist for compounds containing these atoms. Since the mass spectrometer used in this study could not distinguish these isotopomers, the total of the theoretical natural abundances of two isotopomers having the same nominal mass with different formulae was used. For example, the natural abundances for C2 [M-SO 3 ] + were m/z 396.0932 (100.0%), 397.0966 (10.8%), 398.0890 (4.5%), 397.0902 (2.6%), and 398.0975 (1.6%). The theoretical natural abundance for m+1 (m/z 397) was 13.4% and that for m+2 (m/z 398) was 6.1%. Please see Table S-3 for the example of the calculation.