PFOA extraction from the tissue was performed following the method of Mamsen et al., 2017, with some modifications [64 (link)]. Tissues were homogenized at a ratio of 10 parts 70% HPLC grade acetonitrile (in DI water) to one-part tissue using Omni THQ—Digital Tissue Homogenizer for 2 min. Samples were shaken for half an hour at room temperature, then centrifuged at 10,000× g and the clear supernatant containing the extracted PFOA was separated. The supernatant was diluted in 70% acetonitrile in 1:1 and then injected into Ultra Performance Liquid Chromatography–Mass Spectrometry (UPLC–MS) using SYNAPT G2-Si system (Waters Corp., Milford, MA, USA). The mobile phase used was solvent A: 5 mM Ammonium acetate (NH4OAc·H2O) and solvent B: Acetonitrile. The mobile phase was kept at 100% for 2 min, and 98% B for the next 3 min. The column was then conditioned at 100% solvent A for the last 2 min. Calibration curve was built using known concentrations of PFOA (0.78125, 0.390625, 0.195313, 0.097656, 0.048828, 0.024414, 0.012207, and 0.006104 μM) in 70% acetonitrile. Electrospray ionization (ESI) in the mass spectrometry was set in negative ion mode for PFOA detection.
Synapt g2 si system
Manufactured by Waters Corporation
Sourced in United States
The SYNAPT G2-Si system is a high-resolution mass spectrometer designed for advanced analytical applications. It provides accurate mass measurements and high-quality data for a variety of sample types. The system features ion mobility separation technology, enabling the separation and identification of complex molecules based on their size, shape, and charge.
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Lab products found in correlation
Masslynx version 4, by Waters Corporation (1 mentions)
Nd yag laser, by Waters Corporation (1 mentions)
Hdimaging version 1, by Waters Corporation (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Dynamx 3, by Waters Corporation (1 mentions)
Plgs 3, by Waters Corporation (1 mentions)
6 protocols using synapt g2 si system
1
PFOA Extraction from Tissue Samples
2
MALDI-MSI Analysis with SYNAPT G2-Si
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A Waters SYNAPT G2 system was used for preliminary experiments before switching to the more sensitive SYNAPT G2-Si system equipped with a prototype uMALDI source, both provided with a Nd:YAG laser (Waters Corporation, Manchester, UK) (for more information about the uMALDI source, see Barré et al. [22 ]). The data acquiring was performed using MassLynx version 4.1 and HDImaging version 1.5 software (Waters Corporation). The measurements were performed in sensitivity mode with a scan rate of 1.0 s per scan. Different mass ranges were acquired: m/z 50–500 in positive ion mode for drug experiments, m/z 100–1200 in positive ion mode for lipid experiments, and m/z 100–2000 in negative ion mode for lipid experiments. The instrument was calibrated with red phosphorus for both positive and negative ion modes before each measurement. The laser fluence was dependent on the matrix used as well as the application type. It varied between 150 and 350 arbitrary units set in the control software, and no absolute fluence measurement was performed. The spatial resolution was 150 μm × 150 μm for the drug experiments and 100 μm × 100 μm for the lipid experiments.
3
UPLC-MS/MS Analysis of Traditional Medicine
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LC separation was performed using an Acquity I-Class UPLC system (Waters, UK) with an Acquity UPLC BEH C18 column (1.7 μm, 2.1×100 mm). The initial column temperature was 40°C. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The injection volume was 5 μl, and the flow rate was 10 μl/min. MS detection was performed using an SYNAPT G2-Si system (Waters). The data acquisition mode was the MSE. ESI-positive and ESI-negative ionization modes were used in this study. The source and reservation temperatures were set at 120°C and 350°C, respectively. As an external standard, the lock mass compound used was leucine enkephaline (556.2771 positive, 554.2615 negative). The following operation parameters were used: ESI-positive capillary voltage, 3 kV; negative capillary voltage, 2 kV; and cone voltage, 30 V. The collision energies were set as 6 eV ramp (trap) for the low-energy scan and a 20–50-eV ramp (trap) for the high-energy scan. MassLynx 4.1 Waters controlled LC-MS data acquisition. Acquisition data processing was performed using UNIFI1.8 with a traditional medicine library.
4
LRRK2 Protein Deuterium Uptake Analysis
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RCKW proteins were expressed and purified from Sf9 cells [39 (link)]. Measurements were performed using a Waters Synapt G2Si system with nanoACQUITY UPLC and H/DX technology. The RCKW concentration was 5 µM in LRRK2 buffer (pH 7.4) containing 20 mM HEPES/NaOH, 800 mM NaCl, 0.5 mM TCEP, 5% Glycerol, 2.5 mM MgCl2, and 20 µM GDP. Deuterium uptake was measured in the presence and absence of kinase inhibitors MLi-2 (50 µM) or Rebastinib (50 µM). Peptide identification and analysis were performed using PLGS 3.0 and DynamX 3.0 (Waters Corporation) as described in previous publication. The HDX-MS data included at least three technical replicates. Data were corrected for back-exchange using a global back exchange correction factor. Deuterium uptake plots were generated using DECA (github.com/komiveslab/DECA), with data fitted using an exponential curve for visualization.
5
PFOS Extraction and Quantification from Kidney
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PFOS was extracted from kidney tissue following the methods of Mamsen et al. [38 (link)] with some modifications. Kidney tissue was mechanically homogenized in 70% acetonitrile with a 1– 10 ratio for 2 min, and samples were incubated at room temperature overnight for maximum extraction. Samples were centrifuged at a maximum speed of 10 min, and the subsequent supernatant was carefully transferred into a glass vial. Meanwhile, standards of PFOS dissolved in 70% acetonitrile with concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 µM were prepared to generate a standard curve. Extracted samples were analyzed using Ultra Performance Liquid Chromatography–Mass Spectrometry (UPLC–MS) SYNAPT G2-Si system (Waters Corp., Milford, MA, USA). The mobile phase was kept at 100% for 2 min, and 98% B for another 3 min. Column was conditioned with solvent A for the last 2 min. Electrospray ionization was set in negative ion mode for PFOS quantification by mass spectrometry.
6
Glycoprotein Fractionation and Analysis
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