Nexera uplc

Soluble sugar (fructose, glucose, sucrose, and sorbitol) contents were evaluated using the UFLC method with evaporative scattering detection (ELSD) [43 (link)]. About 1 g of fresh plant tissue was ground and diluted with deionized water. The extraction was carried out for 4 h at room temperature, centrifuged at 14,000× g rpm for 15 min. A cleanup step was performed before the chromatographic analysis: 1 mL of the supernatant was mixed with 1 mL 0.01% (w:v) ammonium acetate in acetonitrile and incubated for 30 min at 4° C. After incubation, samples were centrifuged at 14,000× g rpm for 15 min and filtered through 0.22 µm PTFE syringe filter (VWR International, Radnor, PA, USA). Analysis was performed on a Shimadzu Nexera UPLC (Kyoto, Japan) system. Separation was performed on a Supelcosil 250 × 4 mm NH₂ column (Supelco, Bellefonte, PA, USA) using 77% acetonitrile as the mobile phase at 1 mL min⁻¹ flow rate. The calibration method (R² < 0.99) was used for each individual sugar quantification (mg g⁻¹ in FW).

Sixty milligrams of florets of PA64S (F) and PA64S (S) at stage 7 were used to dissolve the extracted lipids. A Nexera UPLC (Shimadzu, Kyoto, Japan) system fitted with a Q-Exactive quadrupole-orbitrap mass spectrometer equipped with a heated electrospray ionization (HESI) source (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the lipidomic analyses. The acquired LCsingle bondMS raw data were analyzed by the progenesis QI software (version 2.3, Waters Corporation, Milford, MA, USA). The positive and negative data were combined to obtain combined data that were imported into the SIMCA software package (version 14.1, Umetrics, Umeå, Sweden). The VIP value of the first principal component of the OPLS-DA model was greater than 1, and the p-value of the t test was less than 0.05 as the criteria for screening differential lipids. Each sample was represented by five biological replicates.

Cultivation samples were centrifuged at 1000g for 10 min (Rotanta 460 R, Hettich Zentrifugen, Germany), and the supernatant was purified using Protein A affinity chromatography. Enzymatic digestions were performed using trypsin, according to the protocol described before (Ozohanics et al. 2012 (link); Turiák et al. 2011 (link)). UPLC-MS analysis of the antibody digest was performed on a Nexera UPLC (Shimadzu Corporation) coupled to a high-resolution micrOTOF-Q II mass spectrometer (Bruker Corporation). Chromatographic conditions were the following: reversed-phase column (Aeris Peptide 1.7-μm XB-C18 particles, Phenomenex Inc., USA) and gradient elution (solvent A 0.1 v/v% formic acid in water; solvent B 0.1 v/v% formic acid in 10 % water and 90 % acetonitrile mixture; flow rate 225 μL/min flow rate, column temperature 30 °C). Mass spectrometric conditions were the following: positive electrospray ionization mode (capillary voltage 4.5 kV; dry gas flow rate 12.0 L/min; dry temperature 200 °C; end plate offset 500 V) and scans acquired in the 140–2000 m/z range. The relative abundance of high-mannose glycoforms in the product amount expressed between two sampling events (i.e., two glycoform measurements) was calculated by using the mass balance in Eq. 1 in order to identify links between specific productivity and product quality. $\Delta \text{relative}M5=\frac{M{5}_n\cdot P_n-M{5}_{n-1}\cdot P_{n-1}}{P_n-P_{n-1}}$

Three representative mice in each group were selected to perform lipid metabolomics analysis using ultra-performance liquid chromatography-mass spectrometry (LC/MS) by Shanghai LuMing Biological Technology Co. Ltd. (Shanghai, China). Briefly, samples were separated using a Nexera UPLC (Shimadzu, Kyoto, Japan) and analyzed by mass spectrometry with Q Exactive (Thermo Scientific). Heated electrospray ionization (HESI) was used for detection in the positive and negative ion modes, respectively. Differentially expressed lipid metabolisms were identified using the DESeq R package functions estimateSizeFactors and nbinomTest. A p value of <0.05 and fold change of >2 or fold change of <0.5 were set as the threshold values for significantly differential expression. The KEGG pathway enrichment analysis of the differential expressed lipid metabolisms were respectively performed using R based on the hypergeometric distribution.