Hypersil gold column

Analysis was performed on HPLC-DAD (Ultimate 3000, Thermo Fischer Scientific, USA) using Hypersil GOLD column (5 µm, 250 mm × 4.0 mm, Thermo Fischer Scientific, USA). Each extract was analyzed twice. A gradient consisting of solvent A (H₂O) and solvent B (acetonitrile:H₂O 30:70 v/v) was applied at a flow rate of 0.8 mL/min as follows: 0.14 min 96% A and 4% B, 28.14 min 14% A and 86% B, 32.14 min 14% A and 86% B, 34.14 min 5% A and 95% B, 47.14 min 5% A and 95% B, 48.14 min 96% A and 4% B, and 56.14 min 96% A and 4% B. The column temperature was held at 30 °C and the injection volume was 20 µL. For confirmation of the peaks from HPLC-DAD, individual peaks were subjected to UHPLC-MS/MS analysis.
The amount of GSLs was quantified using a calibration curve of pure desulfosinigrin solution (range from 0.14 to 1.4 mM) and RPFs for each individual dGSL [13 (link)]. RPF values for quantification of dGSLs were as following: RPF 1.0 for 1, 4, and 6 [14 (link)]; RPF 1.07 and 0.95 for 3 and 7, respectively [15 ]; and arbitrary RPF 1.0 for 2 and 5.

Serum BA profiles were detected using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system, as previously described.^{(17 (link),38 (link))} Briefly, 20 µl of mouse serum was diluted 100-fold with ²H-labelled internal standards and 0.5 M potassium phosphate buffer (pH 7.4). The mixture was injected into a Bond Elut C18 cartridge (200 mg; Agilent Technologies, Santa Clara, CA). Target molecules were eluted in water/ethanol (1:9, vol/vol). The eluate was evaporated under nitrogen until dry and then dissolved in 20 mM ammonium acetate buffer (pH 7.5)/methanol (1:1, vol/vol). An aliquot of the resulting sample solution was injected into the LC-MS/MS system for analysis. Chromatographic separation was performed using a Hypersil GOLD column (150 × 2.1 mm, 3 µm; Thermo Fisher Scientific, Waltham, MA). A mixture of 20 mM ammonium acetate buffer (pH 7.5), acetonitrile, and methanol (70:15:15, vol/vol/vol) was used for the initial mobile phase and was gradually changed to 30:35:35 (vol/vol/vol) over 30 min.

For the
pilot study, offline high-pH reverse-phase fractionation was performed
using a Hypersil Gold column (Thermo Scientific, P/N 25002-202130).
The mobile phase A was water adjusted with ammonium hydroxide to pH
10 and B was 10 mM ammonium bicarbonate in 80% acetonitrile (ACN)
adjusted with ammonium hydroxide to pH 10 and a flow rate of 300 μL/min.
The samples were separated into 91 fractions with each fraction collected
every 60 s from the start of the run and using the gradient shown
in Supplementary Data (S1 Data). For the
larger study, offline high-pH reverse-phase fractionation was performed
using an Xbridge BEH C18 column (Waters P/N 186006710). The mobile
phase A was water adjusted to pH 10 with ammonium hydroxide and B
was 90% ACN adjusted to pH 10 with ammonium hydroxide, at a flow rate
of 200 μL/min. Fractions were collected every 60 s from the
start of the run (100 fractions) and then concatenated into 44 fractions
using the gradient shown in Supplementary Data (S1 Data). The samples analyzed by DIA LC–MS/MS were
not processed by offline fractionation.

Strictosidine synthase (STR) activity was measured using a modified protocol of a previous study [46 ,47 (link)]. One gram of PLBs was frozen in liquid nitrogen and ground to a fine powder. Fifty milligrams of polyvinylpolypyrrolidone and 1 mL of extraction buffer (0.1 mM phosphate buffer (pH 6.3), 3 mM ethylenediaminetetraacetic acid (EDTA) and 6 mM dithiothreitol (DTT)) were added. The mixture was then centrifuged at 18,000 rpm for 15 min at 4 °C. The supernatants were collected as protein extracts and stored at −20 °C. Fifty microliters protein extracts were incubated in 100 mM phosphate buffer (pH 6.8) that contained 0.8 mM tryptamine, 2 mM secologanin and 100 mM d-(+)-gluconic acid-δ-lactone (final reaction volume of 200 μL) for 30 min at 35 °C. The reaction was terminated by adding 100 μL of chilled methanol and filtered through a 0.22 µm nylon filter for HPLC analysis. STR enzymatic activity was determined indirectly by the quantity of strictosidine (µg/mL) in the reaction mixture. Strictosidine was analyzed using an Ultimate 3000 (Waters, Miford, MA, USA) HPLC system coupled to a Hypersil Gold column (4.6 mm × 250 mm × 5 μm, Thermo, Waltham, MA, USA). An aliquot of the reaction (10 μL) was directly injected using a solvent gradient of 15% acetonitrile in 0.05% formic acid for 20 min. The absorbance of strictosidine was monitored at 280 nm.

As described in Sokovic Bajic et al.^{22 (link)} the L. brevis BGZLS10-17 strain was incubated in MRS medium supplemented with 0.6% of MSG (Acros organics). The cells were harvested by centrifugation (4500 × g for 15 min at 4 °C) and 1 ml of the supernatant was evaporated up to 200 μl and diluted 2-fold by 7% acetic acid. The samples were then centrifuged at 8100 × g for 15 min at room temperature. The obtained supernatants were used for HPLC analysis^{63 (link)}. The aliquots of 100 μl [bacterial supernatants and GABA standard (Sigma)] were filtrated through 0.22 μm filters and derivatized to phenylthiocarbamyl-GABA^{64 (link)}. The derivatized samples were dissolved in 200 μl of the initial mobile phase, solution A (138 mM sodium acetate, pH 6.3, 6% acetonitrile, 0.05% triethylamine). HPLC separation was performed on the instrument of Thermo scientific 3000 equipped with a Hypersil gold column (Thermo Fisher Scientific, Waltham, MA, United States 150 × 4.6, 5 μm). The elution solvent system comprised of solution A, solution B (acetonitrile) and solution C (water). The elution program is shown in Supplementary Table 1. The amount of GABA production was calculated from the standard curve.

UPLC was carried out on a Waters ACQUITY H-Class UPLC (Waters Corp, Milford, MA, USA) Chromatographic separations were performed on a Thermo Hypersil Gold column (2.1 mm × 100 mm, 1.9 µm). The column oven was maintained at 40 °C and the mobile phases consisted of solvent A [Water + 0.1% formic acid (v/v)] and solvent B [Acetonitrile + 0.1% formic acid (v/v)]. The elution gradient was as follows: 0–0.5 min, B 15%; 0.5–1 min, B 15–20%; 1–6 min, B 20%; 6–13 min, B 20–30%; 13–23 min, B 30–35%; 23–24 min, B 35–38%; 24–27 min, B 38–60%; 27–31 min, B 60–90%; 31–32 min, B 90–15%; 32–34 min, B 15%. The injection volume was 2 μL and the flow rate was 500 μL/min for each run. Methanol was run after every sample as a blank. Next, MS analysis was conducted on a Waters Xevo G2-S QTOF MS (Waters Corp.) in the negative ion mode. The mass spectrometers performed the MS^E acquisition mode with alternative high- and low-energy scans. The operational parameters were set as follows: source temperature, 120 °C; desolvation temperature, 550 °C; cone voltage, 40 V; capillary, 3.0 kV; cone gas flow, 30 L/h; and desolvation gas flow, 800 L/h. Accurate mass measurements were achieved with the use of an automatic calibration delivery system, which contained the internal reference [Leucine-enkephalin, m/z 556.276 (ESI+), m/z 554.262 (ESI-)]. Data were collected from m/z 100 to 2000 [8 (link)].