Synapt g2 quadrupole time of flight mass spectrometer

0.5 µg of total protein was loaded onto a Nano-Acquity system (Waters Corporation) equipped with a Bridged Ethyl Hybrid C18 1.7 µm, 15-cm×150-µm analytical reversed phase column (Waters Corporation) and operated at a column flow rate of 1 µl/min. All samples were measured in triplicate. Apart from the column dimension and flow rate, all other gradient conditions were as detailed earlier [14] (link). Analysis of tryptic peptides was performed using a Synapt G2 quadrupole time of flight mass spectrometer (Waters Corporation, Manchester, UK) with the operating and experimental conditions as previously described [14] (link). Accurate mass precursor and fragment ion LC-MS data were collected in data-independent MS^e mode of acquisition. This method alternates the energy applied to the collision cell of the mass spectrometer between a low and elevated energy state [16] (link), [17] (link). Briefly, the low energy portion of the obtained data sets is typically used for quantification of the proteins, whereas the combined low and elevated energy information are utilized for identification purposes. In both modes of acquisition, mass spectral information was obtained from m/z 50 to 1990 at a resolving power of at least 10,000 full width half maximum.

All solvents were MS purity and were purchased from Merck Chemicals Pty. Ltd. (Germiston, South Africa). HRMS coupled to liquid chromatography (LC-HRMS) was used for wine fingerprinting. The samples were analyzed by Ultra Performance Liquid Chromatography (UPLC, Waters Corporation) equipped with a Synapt G2 quadrupole time-of-flight mass spectrometer (Waters Corporation). The separation was carried out on an Acquity UPLC HSS T3 column (1.8 μm internal diameter, 2.1 mm × 100 mm, Waters Corporation) using 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B) and a scouting gradient over 10 min. Flow rate was 0.3 mLmin⁻¹ and the column temperature 55 °C. The injection volume was 2 μL and the samples were injected directly without pre-treatment. Mass calibration was performed according to the manufacturer’s procedure. The MS was operated in both positive and negative mode, and the total number of features acquired as RT_m/z was 1466 for each sample. The software is directly integrated with SIMCA-P (SIMCA 14.1, Umetrics, Sweden) and the statistical algorithms are directly applied to the processed datasets [34 (link)].

The process described in Yang et al. and Lin et al. [6 (link),46 (link)] consisted of three steps. (1) The third passage of iPSC-derived RPE cell lines were treated with and without A2E. Three biological replicates were prepared representing three separate cultures derived from each cell line and were also performed separately for A2E-treated samples. (2) Proteins were extracted from each cell line, reduced and alkylated before tryptic digestion, and RapiGest was cleaved with acid. The resulting peptides were analysed using a Synapt G2 quadrupole-time-of-flight mass spectrometer (Waters Corp.) with MSE data-independent scanning. (3) Initial data were processed using ProteinLynx Global Server (Version 2.5 RC9, Waters Corp.). Further analysis was performed with TransOmics software (Waters Corp.) and the NCBI database of human sequences.

The melting points of the prepared compounds were recorded on a Thermocouple digital melting point apparatus and are uncorrected. Their IR spectra were recorded as powders by using the thin-film method on a Bruker VERTEX 70 FT-IR Spectrometer (Bruker Optics, Billerica, MA, USA) equipped with a diamond ATR (attenuated total reflectance) accessory. The Merck kieselgel 60 (0.063–0.200 mm) (Merck KGaA, Frankfurt, Germany) was used as stationary phase for column chromatography. The ¹H- and ¹³C-NMR spectra of the prepared compounds were obtained as CDCl₃ or DMSO-d₆ solutions using the Agilent 500 MHz NMR spectrometer (Agilent Technologies, Oxford, UK) and the chemical shifts are quoted relative to the TMS peak. The low- and high-resolution mass spectra were recorded at the University of Stellenbosch using a Waters Synapt G2 Quadrupole Time-of-flight mass spectrometer (Waters Corp., Milford, MA, USA) at an ionization potential of 70 eV. The synthesis and analytical data of the 2–(4-halogenophenyl)-4-chloroquinazolines 9a and 9 b have been described before¹⁸ (link). Copies of ¹H- and ¹³C-NMR spectra of compounds 6a–e, 7a–e, 8a–e, 10a–j are included as Supplementary Material.

The individual phenolic compounds in the BWEP were analyzed using liquid chromatography-mass spectrometry (LC-MS/MS) following a previously described method (37 (link)). A Waters Synapt G2 quadrupole time-of-flight mass spectrometer (Milford, MA, United States) was used for profiling, along with a Waters Acquity UPLC and a Waters HSS T3 column (2.1 × 100 mm, 1.7 m). The flow rate and injection volume of the solvent A (0.1% formic acid) and solvent B (0.1% acetonitrile) mobile phases were 0.3 mL/min and 2 L, respectively. The gradient elution followed the conditions: 0 min: 100% A, 1–22 min: 28% B, 50 s: 40% B, and 1.5 min: 100% B. This was further re-equilibrated for 5 min. A stock solution containing pure standards (0.1–50 mg/mL), catechin, ellagic acid, epicatechin, gallic acid, punicalagin, punicalagin and chlorogenic acid, rutin, syringic acid, and quercetin was used to determine the structures and quantitative analyses of the phenolic compounds. This was done using calibration curves and the structure-related target analyte/standard (chemical structure and or functional group) principle. For the regression coefficient, good linearity (R² > 0.990) was obtained. The MassLynx 4.1 software was used to collect and process the data, and a metabolomic method was employed to highlight the significant and minute differences and similarities.