Q tof micro mass spectrometer

For the MS/MS analysis of the aspirin acetylated and untreated TrxR1, gels were loaded with 0.31 ug of purified TrxR1 protein. Following SDS-PAGE, a 1 mm circular slice was picked from the gel using an Ettan Robotic Spot-Picker and submitted for proteolytic digestion (Trypsin) and peptide extraction at the Functional Proteomics Facility at the University of Western Ontario. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) analyses of TrxR1 and aspirin-acetylated TrxR1 were performed at the Biological Mass Spectrometry Laboratory at The University of Western Ontario. Gel slices were de-stained with 50 mM ammonium bicarbonate (09830, Sigma) and 50% acetonitrile (00687, Sigma). The protein samples were reduced with 10 mM dithiothreitol (BP25641, Fisher), alkylated with 55 mM acrylamide (BP1406-1, Fisher), and digested with 5 ng/ μl Trypsin (Promega). LC-MS/MS was performed using a Q-Tof Micro mass spectrometer (Waters) equipped with a Z-spray source in positive ion mode (+ 0.1% formic acid) or using Thermo Scientific LTQ-Orbitrap XL mass spectrometer. The data were analyzed and visualized using PEAKS software (Bioinformatic Solutions, Inc, Waterloo, Ontario).

Dried extract (3 mg) of algae was dissolved in 1 mL of MeOH/H₂O 1/1 v/v. The analysis of compounds from algae was carried out with the use of an ACQUITY Ultra Performance LC system equipped with a photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, USA) series with a mass detector Q/TOF micro mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source operating in negative mode at the following conditions: capillary voltage, 2300 kV; source temperature, 100 °C; cone gas flow, 40 L/h; desolvation temperature, 500 °C; desolvation gas flow, 11,000 L/h; and scan range, m/z 50–1500. Separation of individual compounds was carried out using an ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA) at 40 °C. The elution gradient test was carried out using water containing 1% acetic acid (A) and acetonitrile (B), and applied as follows: 0 min, 1% B; 2.3 min, 1% B; 4.4 min, 7% B; 8.1 min, 14% B; 12.2 min, 24% B; 16 min, 40% B; 18.3 min, 100% B, 21 min, 100% B; 22.4 min, 1% B; 25 min, 1% B. The sample volume injected was 2 μL and the flow rate used was 0.6 mL/min. The compounds were monitored at 280 nm. Integration and data elaboration were performed using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) [54 (link)].

All chemicals and reagents were purchased from commercial sources. Organic solutions were concentrated in a rotary evaporator (Bu¨chi Rotavapor) below 55 °C under reduced pressure. Silica gel thin-layer chromatography was performed on precoated plates GF-254 and visualised under UV light. Melting points were determined with a Melt-Temp II apparatus. ¹H NMR and 13C NMR spectra were recorded on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane as internal standard. Chemical shifts are given in ppm (δ). The multiplicities are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartette; m, multiplet; br s, broad singlet. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film. ESI-mass and high-resolution mass spectra (HRMS) were recorded on a Water Q-Tofmicro mass spectrometer. Analytical results are within 0.40% of the theoretical values. Purity of the compounds was analysed by HPLC (Agilent Technologies 1260 Infinity) using 80:20 MeOH/H₂O as the mobile phase with a flow rate of 0.8 ml/min on a C18 column. All tested compounds exhibited greater than 95% purity unless otherwise noted.