Unifi v1

UPLC was performed on a Waters (Milford, MA, USA) ACQUITY UPLC system using an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm; Waters) maintained at an oven temperature of 40 °C. The mobile phase, comprising solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid with acetonitrile), was delivered at a flow rate of 0.5 mL/min. The elution gradient was set as follows: 0–5 min, 3 % phase B; 5–16 min, 3–100 % phase B; 16–17 min, 100 % phase B; 17–19 min, 100–3 % phase B; and 19–20 min, 3 % phase B. MS detection was carried out using a SYNAPT G2-Si HDMS QTOF mass spectrometer (Waters) with electrospray ionization. The MS detector conditions were as follows: ESI-positive capillary voltage, 3 kV; negative capillary voltage, 2 kV; cone voltage, 40 V; source temperature, 120 °C; and desolvation temperature, 500 °C. The MS/MS data were obtained using a collision energy ramp from 20 to 40 eV in the MS^E mode. The scanning time was 0.2 s, with a mass range m/z of 50–1200 Da. A solution of leucine encephalin, sprayed at a flow rate of 10 μL/min, served as a reference ion for both positive (m/z 556.2771) and negative (m/z 554.2615) ion modes. Data acquisition and analysis were managed using the UNIFI V1.71 software (Waters). Identification of peaks was carried out by screening against the propriety scientific library of UNIFI V1.71.

Unbiased metabolomics analysis was performed as previously described using an ultra-performance liquid chromatography (UPLC) system (Waters, Milford, USA) [41 (link)]. The scan range in MS and MS/MS modes included 50–1200 m/z. Data acquisition and analysis were controlled by Waters UNIFI V1.71 software. The metabolic profile of the TLC-purified pigment from the transformed S. cerevisiae was compared with that of the purified phleichrome from C. phlei obtained in our previous studies [5 (link), 38 (link), 39 (link)].

UPLC-Q/TOF–MS analysis was performed on a Waters Xevo G2-XS quadruple time-of-flight spectrometer (Waters, Milford, MA, USA) coupled with UNIFI v1.7.1 software (WatersCorp., Milford, MA, USA). Chromatographic separation was performed with the flow rate of 0.2 mL/min on an Acquity UPLC BEH C18 Column (2.1 mm × 100 mm, 1.7 μm; Waters) and C18 Pre-column (2.1×5 mm, 1.7 μm; Waters) at 25 °C. The mobile phase used was a mixture of acetonitrile (A) and 0.1% formic acid water (B). The gradient elution as follows: 0–5 min, 70–60% B; 5–10 min, 60–40 % B; 10–15 min, 40–40% B; 15–20 min, 40–34% B; 20–22 min, 34–34 % B; 22–24.5 min, 34–25% B; 24.5–27 min, 25–15% B; 27–29 min, 15–15% B; 29–31 min, 15–70% B; and 31–33 min, 70–70% B. The injection volume of the sample was 1 μL. Mass spectra were recorded over the range of m/z 50–1200 under the following conditions: capillary source of 3 kV, sampling cone source of 40, source offset of 80, source temperature of 110 °C, cone gas flow rate of 50 L h⁻¹, and desolation gas flow rate of 600 L · h⁻¹. Leucine enkephalin was used to calibrate the mass spectrometer.