Human and mouse sEPCR were expressed in Drosophila melanogaster S2 cells and purified by nickel-nitrilotriacetic acid affinity chromatography and MonoQ ion-exchange chromatography (18 (link)). Purified protein was mixed with LBPA, repurified on MonoQ, and lipid exchange confirmed on NativePAGE 4 to 16% Bis-Tris gels. Lipid loading was quantified by extracting sEPCR solutions with methanol (1:3 v/v) followed by injection onto an Acquity UPLC system with a Waters Acquity BEH column. LBPA and phosphatidylcholine were quantified after specific fragmentation on a Waters XEVO TQ-XS tandem mass spectrometer. aPL HL5B binding was measured by surface plasmon resonance after capture of human sEPCR on a lipid bilayer made of PC-SMA-PG-cholesterol-DOGS (50–20-5–20-5) immobilized on L1 chip surface. Presented experimental data are representative of three independent repeats and were fitted with the BIAevaluation3.2 package using global fitting and comparing a Langmuir 1:1 model with a bivalent analyte model.
Xevo tq xs tandem mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The XEVO TQ-XS is a tandem mass spectrometer designed for high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) applications. It provides accurate and precise quantitative and qualitative analysis of small molecules.
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Lab products found in correlation
4 protocols using xevo tq xs tandem mass spectrometer
1
Purification and Lipid Binding of sEPCR
2
UPLC-MS/MS method for analyte analysis
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All analyses were conducted using a Aquity I‐Class ultra‐performance liquid chromatography (UPLC) system (Waters, Eschborn, Germany) interfaced via unispray (US) ionization to a Xevo TQ‐XS tandem mass spectrometer (Waters). The LC system was equipped with a Poroshell C‐8 analytical column (50 × 3.0 mm, 2.7 μm particle size; Agilent, Waldbronn, Germany). The LC method employed 10 mM aqueous ammonium acetate (solvent A) and acetonitrile (solvent B) and gradient elution starting with 95% A, decreasing to 0% A in 10 min, maintaining 0% A for 2 min before re‐equilibration at 95% A for 2.5 min. The US source was operated in positive mode using an impactor voltage of 1 kV and the mass spectrometer recorded two diagnostic precursor/product ion pairs per analyte in multiple reaction monitoring (MRM) mode. The collision gas was nitrogen (provided by a nitrogen generator, CMC, Eschborn, Germany), and collision energies were optimized for each ion transition as summarized in Table 1 .
3
Tobacco Exposure Biomarker Quantification
4
UPLC-MS/MS Quantification of Analytes
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Chromatographic separation was performed using an ACQUITY® UPLC® H-Class UPLC system (Waters, USA) with a Kinetex Biphenyl LC column (100 mm × 2.1 mm, 2.6 μm, Phenomenex) preceded by a Security Guard™ ULTRA Cartridge UHPLC Biphenyl 2.1 mm ID column. The mobile phases comprised solvents A and B: 2% and 99.9% methanol in 0.1% formic acid solution, respectively. The gradient elution profile is shown in Table 1 . The analytes were then quantified using a Xevo® TQ-XS tandem mass spectrometer (Waters Corporation, Milford, MA US). The mass spectrometer was operated in multiple reaction monitoring modes (MRMs). The MRM transitions and conditions for the analytes and ISs, as well as the retention times, target ions, and qualifier ions used for identification and quantification, are shown in Table 2 .
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