Inertsustain aq c18 column

Recombinant CWPO-C (rCWPO-C) was prepared and purified according to the described methods by Shigeto et al. (2012 (link)). The oxidation of IAA by rCWPO-C was carried out in a 100 mM sodium acetate buffer (pH 5) containing 500 µM H₂O₂, 500 µM IAA, 2 U/ml rCWPO-C and rotary-incubated (Rotaflex, Argos) at 10 rpm for 0, 60, 120, 360 min at 25 °C. One U of CWPO-C oxidation activity was defined as the formation of the oxidation product from 2,6-dimethoxyphenol (2,6-DMP) in one µmol/min (Shigeto et al. 2012 (link)). As control, the reaction was carried out in the absence of rCWPO-C. Reaction mix was filtered with a Minisart SRP 4 PTFE-membrane and an aliquot of reaction mixture (20 µl) was analyzed by reverse-phase HPLC on a InertSustain AQ-C₁₈ column (100 nm, 5 μm, 4.6 × 150 mm; GL Sciences, Japan) using an isocratic elution buffer containing methanol : 1% formic acid mixture (40:60, v/v) at a flow rate of 1.0 ml/min. The eluted products were monitored at the absorbance of 250 nm using a Jasco UV-2070 Plus detector.

The reaction mixtures contained 100 μL buffer (100 mM triethylamine phosphate, pH 8.0), 0.1 mM NAD⁺, 10 mM UDP-GlcA, and 0.48 mg of UAXS. A total of 30 parallel tubes were used. The reactions were performed at 25 °C for 4 h and then centrifuged at 21,130 × g for 30 min. The products were subsequently purified by reversed-phase HPLC. HPLC was performed on an Inertsustain AQ-C18 column (5 μm, 4.6 × 250 mm; GL Sciences, Tokyo, Japan) at a flow rate of 1.0 mL/min. The mobile phase was a gradient elution of solvents A (100 mM N,N-dimethylcyclohexylamine phosphate buffer, pH 6.5) and B (30% (v/v) ACN). A gradient elution program was used: 0 min, 100% A; 13 min, 100% A; 35 min, 33% A; 39 min, 33% A; 40 min, 100% A. The eluted fractions were monitored by measuring the UV absorbance at 262 nm (Supplementary Fig. 156). After freeze-drying, UDP-apiose was dissolved with triethylamine phosphate for use.

The procedures for LC–MS, including analysis, peak detection, alignment, and annotation, were described elsewhere [29 (link)]. Briefly, leaf samples were prepared using methanol and MonoSpin M18 columns (GL Sciences, Tokyo, Japan). Samples were analyzed using a SHIMADZU Nexera X2 high-performance liquid chromatography (HPLC) instrument (Kyoto, Japan) with an InertSustain AQ-C18 column (2.1 × 150 mm, 3 μm particle size) (GL Sciences) connected to a Thermo Fisher Scientific Q Exactive Plus high-resolution mass analyzer (Waltham, MA, USA).
The LC–MS data obtained above were converted to mzXML format using ProteoWizard (Palo Alto, CA, USA). Peak detection, determination of ionizing states, and peak alignments were performed automatically using the data analysis software PowerGetBatch developed by the Kazusa DNA Research Institute [42 (link),43 ]. The exact mass values of the nonionized compounds calculated from the adducts were used to search candidate compounds against the UC2 chemical mass databases [44 (link)] (i.e., a combination of two databases, KNApSAcK [45 (link)] and the Human Metabolome Database [46 (link),47 (link)]) with the search program MFSearcher [48 (link)]. The LC–MS results were compiled in the Microsoft Excel file “LCMS_Result Field Data KDRI” (Supplementary File S1).

Metabolite extraction and metabolome analysis were conducted at Kazusa DNA Research Institute. Briefly, 100 mg FW of Arabidopsis shoots were extracted with 75% methanol, loaded on a MonoSpin C18 column (GL Science), and eluted with 75% methanol. Three biological replicates for each treatment were used for analysis. The analysis was performed using a high-performance liquid chromatography (HPLC) Ultimate 3000 RSLC (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a high-resolution mass spectrometer Q Exactive (Thermo Fisher Scientific) with electrospray ionization (ESI) in the positive mode. Chromatographic separation was achieved using an Inert Sustain AQ-C18 column (2.1 mm × 150 mm, 3 µm-particle, GL Science). The column was kept at 40 °C, and the flow rate was 0.2 ml min⁻¹. The mobile phase solutions were water with 0.1% formic acid (eluent A) and acetonitrile (eluent B) and were implemented in the following gradient: 0–3 min, 2% B; and 3–30 min, 2–98% B. The injection volume was 2 µl. Mass spectrometry conditions were as follows: the scan range was set at m/z 80–1200. The full scan resolution was 70,000. The MS/MS scan resolution was 17,500. The obtained data was analyzed using a ProteoWizard (http://proteowizard.sourceforge.net) and a PowerGetBatch (Kazusa DNA Research Inst.). Then, the KEGG database (http://www.genome.jp/kegg/) was used to annotate the metabolites.

Chromatographic separation was done at 40°C using an InertSustain AQ‐C18 column (3 µm HP, 150 mm × 3 mm; GL Sciences, Tokyo). The mobile phase consisted of sodium acetate buffer (50 mM, pH 3.6):acetonitrile (77:13, v/v). The flow rate was 0.9 mL/min. The detector wavelength was set at 307 nm.

An Agilent 1290 Infinity UHPLC system (Agilent Technologies, Tokyo, Japan) was used for LC, and an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies) was used for MS. A 100 mm × 3.0 mm InertSustain AQ-C18 column with a particle size of 1.9 μm (GL Science, Tokyo, Japan) was used, and the temperature was set to 60 °C. Distilled water (DW) containing 0.1% formic acid was used for mobile phase A, and acetonitrile containing 0.1% formic acid was used for mobile phase B. The analytes were separated at a flow rate of 0.8 mL/min. The gradient was started at 10% B, increased linearly to 95% B from 0.5 min to 4 min, maintained at 95% B for 1 min, and then returned to 10% B and equilibrated for 0.5 min before the next injection. The injection volume was 20 μL, and electrospray ionization (ESI) was performed in positive mode. The retention time (RT), multiple reaction monitoring (MRM) transition, and collision energy (CE) for each analyte are listed in Table 2.

Pigments were extracted from ∼10 mg wet cell weight of GF1 grown on marine agar plates with methanol using a Bioruptor UCD-250 sonicator (Cosmo Bio, Tokyo, Japan) for 10 s. The LC-MS/MS analysis was performed using LC (Nexera System; Shimadzu, Kyoto, Japan) triple quadrupole mass spectrometry (LCMS-8060; Shimadzu). The samples were separated by an InertSustain AQ-C₁₈ column (150 mm by 2.1 mm inside diameter [i.d.] and 1.9-μm particle size; GL Sciences, Osaka, Japan) at a column temperature of 35°C. The mobile phase comprised 90% (vol/vol) methanol containing 0.1% (vol/vol) formic acid at a flow rate of 0.4 ml/min. Electrospray ionization was performed at 4 kV, with the positive mode at 250°C in the desolvation line and 300°C in the interface. Nebulizing and drying gases were set at flow rates of 2 and 10 liters/min, respectively. Data acquisition was performed based on the MRM mode (Table S1), and the zeaxanthin abundance was determined on the basis of the peak area of the transition from m/z 568.30 to 476.35. A zeaxanthin standard was purchased from Sigma-Aldrich.