Dionex ultimate 3000 uplc system

Crocin I and crocin II were purchased from the National Institutes for Food and Drug Control (China). Acetonitrile and methanol were of chromatographic purity, and all of the other solvents were analytically pure. The red fruits, green fruits and leaves with three repetitions were freeze dried and then ground to a powder for each sample. The powder of each sample (0.25 g) was accurately weighed and extracted with 50% methanol to a final volume of 25 mL. After treatment with an ultrasonic instrument for 45 min, 50% methanol was added to complement the weight loss and the sample was then filtered. The filtrate was further passed through a 0.22 μm membrane filter to yield the test sample solution. Moreover, the mixed standard solution that included 0.035 mg/ml crocin I and 0.015 mg/ml crocin II was prepared by dissolving the sample in methanol. The chromatographic analysis was performed on a DIONEX Ultimate 3,000 UPLC system with a DAD detector (Thermo Fisher Scientific, USA). The UPLC separation was performed on Waters BEH-C18 column (100 × 2.1 mm, 1.7 μm), operated at 35°C. The mobile phase was acetonitrile (A) and H₂O (B) with the flow rate of 0.3 mL/min. The injection volume was 2 μL and the mobile phase gradient was as follows: 0–6 min, 5–12% A; 6–25 min, 12–48% A. The detection wavelength of crocins was 440 nm.

Sample preparation and metabolites/lipid extraction and identification were performed as described previously (44 (link), 45 (link)). For metabolomic analysis, fresh mycelia were extracted using MeOH:water (1:1). Extracts were dried in a vacuum centrifuge, resuspended in MeOX-pyridine and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylsilyl chloride (TMCS) for derivatization, and analyzed by GC-MS (Thermo Fisher Scientific, Boston, MA, USA) equipped with an RTX-5MS column (30 m by 0.25 mm by 0.25 μm, Restek, Bellefonte, PA, USA). For lipidomic analysis, freeze-dried mycelia were extracted with methyl tert-butyl ether. The lipidomic analysis was conducted using a Dionex UltiMate 3000 UPLC system (Santa Clara, CA, USA) coupled to a HESI probe with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher, CA, USA). The “raw” format files were converted to “abf” format using the ABF converter. The MSDIAL4.20 equipped with the DB_FiehnBinbase-FiehnRI and LipidMsmsBinaryDB-VS46-FiehnO database was used for metabolite and lipid molecule identification, respectively (46 (link)). All annotations were manually checked again. The mass spectrometry data have been deposited to MetaboLights with the data set identifier MTBLS4103.

Samples were loaded with buffer A (0.1% FA in water) onto a 50 cm EASY-Spray column (75 µm internal diameter, packed with PepMap C18, 2  µm beads, 100 Å pore size) connected to a nanoflow Dionex UltiMate 3000 UPLC system (Thermo) and eluted in an increasing organic solvent gradient from 4% to 28% (B: 98% ACN, 0.1% FA, 2% H₂O) at a flow rate of 300 nl/min. Mass spectra were acquired with an orbitrap Fusion Lumos mass spectrometer (Thermo) in the data-dependent mode with MS1 scan at 120,000 resolution and MS2 at 50,000 (@200 m/z) in the mass range from 400 to 1600 m/z. Peptide fragmentation was performed via higher-energy collision dissociation (HCD) with energy set at 35 NCE.

A Dionex Ultimate 3000 UPLC System (Thermo Fisher Scientific, Waltham, MA, USA) coupled in series to a UV detector, a 3-inch NaI(Tl) radioactivity detector, and an ultra-high resolution time-of-flight mass spectrometer with electrospray ionization (ESI) (MaXis Impact, Bruker, Bremen, Germany) was used for analysis of all non-radioactive DV1-k-(DV3) constructs. Solvent A (water, 0.1% HCOOH) and solvent B (acetonitrile, 0.1% HCOOH), flow rate 0.6 mL/min, Acquity UPLC BEH C₁₈ 1.7 µm 2.1 × 50 mm column (Waters Corporation, Milford, MA, USA). The elution gradient was: 0–2 min: 95% A; 2–10 min: from 95% A to 5% A; 10–12 min: 95% A. UV monitoring of the eluate was performed at 220 nm. For deconvolution analysis of the raw mass spectral data, the software program DataAnalysis (Bruker Daltonik, Bremen, Germany) was used. Calculated average neutral molecular ion mass values were obtained using Compass IsotopePattern (version 3.2, Bruker Daltonik, Bremen, Germany) software.

Thermo Fischer TSQ Quantum Ultra AM mass spectrometry system (equipped with ESI source) and Dionex Ultimate 3000 UPLC system (Thermo Fischer Scientific Inc., Waltham, MA); Sartorius BS21S and BS110S electronic balance (Sartorius Scientific Instruments Corporation, Beijing, China); TGL-16 refrigerated centrifuge (Xiangyi Instrument Corporation, Changsha, China); RE-207B rotary evaporator (Keer Equipment PTY., Ltd., Nanjing, China).