E2695 system

Melting points were measured with a WRS-1B capillary melting point apparatus. Optical rotations were obtained with a Rudolf Autopol IV at 22 °C. UV and IR spectra were recorded on a Hitachi U-2900E UV spectrophotometer and a Thermo Scientific Nicolet Is5 FT-IR spectrometer, respectively. ECD spectra were recorded on a JASCO-810 spectropolarimeter. ESI-MS and HRESIMS were acquired on an Agilent 1100 LC/MSD mass spectrometer and an AB Sciex Triple TOF 5600 spectrometer, respectively. X-ray crystallographic data were measured on a Bruker Apex Duo Diffractometer (Ga Kα). 1D and 2D NMR spectra were performed on a Bruker Avance III 400 MHz or a Bruker Avance III 600 MHz spectrometers using the residual solvent signals as the internal standard. All chemical shifts were expressed in ppm. Semi-preparative HPLC was performed on a Waters e2695 system coupled with a 2998 photodiode array (PDA) detector and a 2424 evaporative light-scattering detector (ELSD). A SunFire C18 column (5 μM, 10 × 250 mm; flow rate: 3.0 mL min⁻¹) and a X-bridge C18 column (5 μM, 10 × 250 mm; flow rate: 3.0 mL min⁻¹) were utilized. Thin-layer chromatography (TLC) was performed on pre-coated plates (GF₂₅₄, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China). TLC spots were visualized under UV light (254 or 365 nm) and by spraying with 5% H₂SO₄/vanillin followed by heating to 120 °C.

Chromatographic analysis was carried out to evaluate the levels of two kinds of GnRH regulatory neurotransmitters GABA and glutamate in the brain/pituitary. Weigh the brain and add in the mix of methanol and water (1:1) at the ratio of 100 mg/ml to prepare homogenate. The brain homogenate was centrifuged at 12000 rpm for 30 min at 4 °C. The mix of supernate and 80 μL acetonitrile were centrifuged at 12000 rpm for 30 min at 4 °C. The new supernate was derivatized, mixed with 80 μL borate saline buffer, 160 μL water and 80 μL F-moc, filtered by 0.45 μm filtration membrane and immersed in 40 °C bath for 5 min. Then, the samples were used for the measurement of GABA and glutamate using chromatographic analysis. All standard biomarkers used for identification purpose in chromatographic studies were procured from Zhongke Co, Ltd., Beijing, China. High-performance liquid chromatography (HPLC) was performed on a Waters e2695 system (Waters, USA) equipped with a hypersil ODS column (Elite, Dalian, China). The mobile phase A was a mix of natrium aceticum (pH 4.8),water and tetrahydrofuran with the ratio of 410:85:5, and mobile phase B was pure acetonitrile. Flow rate was 1 ml/min. The column temperature was set at 30 °C. Samples were detected at the wavelength of 265 nm. Injection volume was 20 μL.

To determine peptide hydrophobicity, the Waters Alliance e2695 system with a Waters 2998 PDA Detector (software-Empower^®3) was used. All analyses were carried out on a Phenomenex Luna C18(2) column (3.0 × 100 mm, 5 µm particle size, 100 Å pore size). The peptides were dissolved in water (0.1% TFA, v/v) to obtain a concentration of 1 g/L. UV detection at 214 nm was used, and aliquots (10 µL) were eluted with a linear 10–55% acetonitrile gradient in deionized water over 60 min at 25.0 ± 0.1 °C. The mobile phase flow rate was 0.5 mL/min. Both eluents contained 0.1% (v/v) of TFA. Each peptide sample was analyzed in triplicate.

The linkages and hydroxyl groups of the fractionated lignin from corn stover biomass were analyzed using 2D NMR and ³¹P NMR (Liu et al., 2019a (link); Meng et al., 2019 (link); Xu et al., 2022 (link)). The lignin molecular weight distribution before and after fermentation was analyzed by gel permeation chromatography (GPC). The analysis was carried out on a Waters e2695 system equipped with a variable 2489 UV/Vis detector at 280 and 254 nm and two tandem 30 cm × 7.8 mm TSKgel GMPWxl column (TOSOH, Tokyo). As alkaline solution can dissolve the lignin, 0.1 M NaOH was employed as a mobile phase with a flow rate of 1.0 ml/min (Liu et al., 2017 (link); Xu et al., 2022 (link)).

Purifications were carried out on a Phenomenex Gemini-NX C18 column (21.20 × 100 mm, 5.0 µm particle size, 110 Å pore size). UV detection at 214 nm was used, and the crude peptides were eluted with a linear 10–70% acetonitrile gradient in deionized water over 90 min at room temperature. The mobile phase flow rate was 10.0 mL/min. Acetonitrile and water, both containing 0.1% of TFA, were used as a mobile phase. The purity and identity of the peptide was confirmed with LC-MS analysis. The RP-HPLC system was used—Waters Alliance e2695 system with Waters 2998 PDA and Acquity QDA detectors (software—Empower®3). All analyses were carried out on a Waters XBridge™ Shield RP-18 column (4.6 × 150 mm, 3.5 µm particle size, 130 Å pore size). Samples (10 µL) were analyzed with a linear 10–90% acetonitrile gradient in deionized water over 15 min at 25.0 ± 0.1 °C. The mobile phase flow rate was 0.5 mL/min. Both eluents contained 0.1% (v/v) of formic acid. Mass analysis and UV detection at 214 nm were used. Pure fractions (>95%, by HPLC analysis) were collected and lyophilized.