Xevo g2 xs qtof

Due to the deceptive substrate-dependent NADH oxidase activity of the flavoprotein component, 4-HPA 3-hydroxylase could not be assayed either by monitoring the substrate-dependent oxidation of NADH or by monitoring O₂ consumption [15 (link)]. Hence, to demonstrate that 3,4-DHPA was the intermediate product of 4-hydroxyphenylacetic acid (4-HPA) degradation by 4-HPA 3-hydroxylase, DHPAO was utilized for the transformation 3,4-DHPA into 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS). The characteristic yellow color product of CHMS presented a maximum absorption at 380 nm [27 (link)]. With a saturating amount of DHPAO, the rate of formation of CHMS represents the rate of formation of 3,4-DHPA due to 4-HPA 3-hydroxylase activity. The reaction mixture (2 mL) contained 50 mM phosphate buffer (pH 8.0), 280 μM NADH, purified HpaB, HpaC and MhpB2 to final concentrations of 40.1, 25.5 and 21.0 μg/mL, respectively. The reaction was initiated by the addition of 165 μM 4-HPA at 25 °C and sustained for 30 min. In order to further confirm the conversion of 4-HPA to 3,4-DHPA by 4-HPA 3-hydroxylase, reaction was carried out without the addition of DHPAO and the reaction mixture was detected by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis which was performed on a quadrupole-time of flight mass spectrometer (Waters Xevo G2-XS QTof, USA).

Lipid extracts (n = 10) were reconstituted in 990 µL MeCN:IPA (1:1 v/v) briefly vortex mixed, and centrifuged for 15 min ca 16,000 × g. 100 µL supernatant was transferred to a total recovery vial (Waters Corporation, USA). Mass spectrometric analysis was performed using an Acquity I-Class UPLC system (Waters Corporation, USA) fitted with a 2.1 × 100 mm CSH C18 analytical column heated to 55 °C. Lipids were separated by gradient separation as shown in Table 1. Mobile phase A was 10 mM ammonium formate 60:40 acetonitrile:water with 0.1% formic acid and mobile phase B was 10 mM ammonium formate 90:10 propan-2-ol:acetonitrile with 0.1% formic acid.Table 1

Gradient conditions for LC–MS analysis.

Time (min)	Flow rate (mL/min)	% A	Curve
0	0.4	60	–
2	0.4	57	6
2.1	0.4	50	1
12	0.4	46	6
12.1	0.4	30	1
18	0.4	1	6
20.4	0.4	1	6
20.5	0.4	60	1
22.5	0.4	60	1

Online MS analysis was performed using a Xevo G2-XS Q-ToF (Waters Corporation, Manchester) in positive ion sensitivity mode over the m/z range 120–1200.

Samples were collected at 10 and 15 DPA grains of bg1 and WT for metabolome sequencing analysis, which were consisitenti with samples used in the RNA-seq analysis. Samples were ground to powder using a grinder (MM 400, Retsch) and dissolved into extraction solution to extract by ultrasonic extraction. The extracted metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with Waters Xevo G2-XS QTOF. The metabolomic experiments and conjoint Analyses of transcriptome and metabolome sequencing were conducted by BMKcloud, Beijing, China (http://www.biomarker.com.cn/) following the manufacturer’s instructions.

All chemicals and solvents were purchased from Sigma-Aldrich, Bangalore, India and TCI chemicals, Hyderabad, INDIA. Pre-coated silica gel TLC plates monitored the completion of the reaction. ¹H and ¹³C NMR were recorded on an Agilent (400 MHz), and JEOL ECZ500R/S1 NMR spectrophotometer (500 MHz); chemical shifts are expressed as ppm. TMS and CDCl₃ were used as internal standards and solvents. LC-MS was recorded on a Waters mass spectrometer (Xevo G2-XS QTof) (water column: Sunfire C18, 4.6 × 250 mm).

Optical rotations were measured on a Bellingham Stanley ADP 400 Polarimeter. UV and IR readings were measured on a PerkinElmer UV-Visible spectrophotometer and a PerkinElmer spectrum 100 FT-IR spectrophotometer, respectively. ¹H, ¹³C, and 2D NMR spectra, including COSY, HSQC and HMBC, were recorded in CDCl₃ on a 400 MHz Bruker NMR spectrometer using the residual solvent signal (δ_H 7.26 and δ_C 77.4) as internal standards. Additional 1D selective NOE and NOESY spectra were obtained on a 600 MHz Bruker NMR spectrometer using the residual solvent signal (δ_H 7.26 and δ_C 77.4) as internal standards. HPLC isolation and purification of 1 was conducted on a Shimadzu LC-8A preparative LC coupled to a Shimadzu SPD-M10A VP diode array detector. Both HRESIMS data and LC-HRMS/MS analyses were obtained on a Waters Xevo G2-XS qTOF with an ESI positive ion mode and data-dependent acquisition mode. An Agilent 1100 series coupled with an Agilent LC/MSD (Liquid Chromatography/Mass Selective Detector) trap XCT mass spectrometer, equipped with an ESI interface system in negative mode, was used for the detection of the L-Marfey’s-derivatized L/D-valine, proline, N-methylphenylalanine, and alanine moieties from benderamide A.

The purity of wild-type and mutant ColH enzymes was first examined by SDS-PAGE. Proteins were separated on a 7.5% polyacrylamide gel and stained with Coomassie brilliant Blue R. Then, the purity was tested by size exclusion chromatography (SEC) and SDS-capillary electrophoresis (CE-SDS). SEC was performed according to USP <89.2> Collagenase II [23 ], and CE-SDS was performed according to Chinese Pharmacopeia 2015. Q-TOF-MS (XevoG2-XS Q-TOF, Waters) was employed to identify the molecular weight (MW) of rColH(E451D) (conducted by Shanghai Applied Protein Technology Co., Ltd.). The test time was 20 minutes, and the positive ion & parent ion scanning range was 500–4000 m/z.