Accela hplc

Isolated lipocalin proteins were dialysed against ultrapure water prior to analysis by MicroTOF-Q mass spectrometry (column Ace C8 50 × 2.1, mass spectrometer Thermo Scientific LTQ Orbitrap XL with an ESI source via a Thermo Scientific Accela HPLC). Mass spectrum peaks were averaged and deconvolution to uncharged neutral mass was performed using Xtract within the Xcalibur software (the BioCentre, University of Reading).

The detection of apocynin in tissue sample was performed as described previously with some modifications.¹⁷ In brief, apocynin was detected using an Accela HPLC coupled to a linear ion‐trap tandem mass spectrometry (LTQ‐Orbitrap XL; Thermo‐Fisher Scientific) (HPLC‐MS/MS) in the negative ion mode. Phenacetin (500 ng/mL) was used as an internal standard in every vial. A reversed‐phase 50 × 2.1 mm ID, 1.9 mm particle, 175 Å pore C18 Hypersil Gold column (Thermo Scientific) was used with injection volume set at 5 µL for the separation of apocynin and phenacetin. Calibration curve used for PK analysis was constructed by spiking apocynin in mouse plasma using eight calibrators ranging from 1 to 10000 ng/mL with great reproducibility and R² ≥ 0.996 for subsequent 5 separate experiments on different days. Best fit curve was achieved using linear regression model (1/ꭓ² weighting factor) which showed minimum percentage relative error. Limit of detection for apocynin was set up at signal to noise ratio ≥ 3,¹⁸ which is 1 ng/mL in our system. The limitation of quantification was set up at signal to noise ratio ≥ 10, which is 10 ng/mL in our system. Orbitrap component was used for the full scan spectra of apocynin and diapocynin.

Maternal serum or venous cord blood samples were prepared as described for the HPLC method and pooled (n = 10). An Accela HPLC (Thermo Fisher Scientific) was used with a TSKgel Amide-80 (Tosoh Bioscience, Japan) and a linear gradient of a 50 mmol/L-ammonium formate/acetonitrile solvent system. Oligosaccharides were determined by a TSQ Quantum Ultra (Thermo Fisher Scientific, Waltham, MA, USA) triple quadrupole instrument in positive ESI mode. The spray voltage was set to 4000 V, capillary voltage to 35 V, and vaporizer temperature was 250 °C. Scan width was 1 Da with 0.1 s scan time for each transition.

Gas in bottles’ headspace was sampled using a gas tight syringe and analyzed for H₂ and CH₄ with a Compact GC^4.0 (Global Analyser Solutions, Breda, Netherlands) equipped with Carbonex 1010 column (Supelco, 3 m × 0.32 mm) followed by a Mosieve 5A column (Restek, 30 m × 0.32 mm) and a thermal conductivity detector (TCD). Argon was used as carrier gas at 0.8 mL min^-1. Standard GC settings for H₂ and CH₄ measurement were: 300 kPa; valve (injection) oven: 60°C; column oven: 100°C; TCD temperature: 100°C; filament: 175°C.
Liquid samples were analyzed for volatile fatty acids (VFA) and alcohols with an Accela HPLC (Thermo Scientific, Waltham, MA, United States) equipped with a Varian Metacarb 67H column (Agilent, 300 mm × 6.5 mm) and a refractive index detector. Column was kept at 45°C and running with 0.01N of H₂SO₄ as eluent at a flowrate of 0.8 mL/min.
Sulfate concentrations were measured in an ICS-2100 Ion-Chromatograph system (Thermo Scientific) equipped with an AS19 column (250 mm × 2 mm) using a hydroxide (gradient) solution as eluent. Sulfide was measured using the methylene blue method, as described by Trüper and Schlegel (1964) (link).

LC-MS data were acquired on an Accela HPLC (Thermo Fisher Scientific) coupled to an Exactive Orbitrap (Thermo Fisher Scientific, Hemel Hempstead, UK) in both positive and negative mode set at 50,000 resolution (controlled by Xcalibur version 2.1.0; Thermo Fisher Corporation, Hemel Hempstead, UK). The mass scanning range was m/z 75–1200; the capillary temperature was 320 °C; and the sheath and auxiliary gas flow rates were 50 and 17 arbitrary units, respectively. The separation was performed on a ZIC-pHILIC column (150 × 4.6 mm, 5 μm from HiChrom, Reading, UK) in binary gradient mode. The mobile phase used was 20 mM ammonium carbonate buffer (pH 9.2) and pure ACN; the flow rate was 300 μL·min⁻¹. The gradient was programed as follows: 0 min 20% A/80% B to time 30 min 80% A/20% B. The injection volume was 10 μL, and the sample tray temperature was controlled at 12 °C during the measurement. Samples were run in a stratified method with between-subject samples placed in randomised order.

Residues in NS5 that contact RNA were mapped using the reversible crosslinking affinity purification assay19 (link). ZIKV NS5 2 μM were mixed with RNA PE46 RNA (4 μM) and crosslinked with formaldehyde and processed for mass spectrometry. Control reactions were processed in parallel in the absence of formaldehyde. HPLC–MS analysis was conducted on an LTQ Orbitrap XL mass spectrometer equipped with an Accela HPLC and an electrospray ion source (Thermo Scientific). Peptides were eluted over a 90-min gradient, and tandem MS data was acquired using collision-induced dissociation. Peptides were identified using SearchGUI (v3.1.0)32 (link), and searched against a concatenated target/decoy database constructed from the cRAP database (http://www.thegpm.org/crap/index.html) of the sequence of ZIKV NS5. Identification settings included an unspecific protease, 10 p.p.m. MS1, and 0.3 Da MS2 error tolerances and oxidation of methionine as a variable modification. MS2 peptide spectrum matches were inferred using PeptideShaker (v1.13.3)33 (link). Posterior error probability was calculated in PeptideShaker using the ratio of hits from the decoy database relative to the true database search. Only assignments with high confidence and in two independent replicates and those absent in the control reactions were used.