RKO and DLD-1 cells were plated at a density of 18,000 cells per well and treated with 10 µM of the NAT2-specific substrates amonafide (Adooq Bioscience) and procainamide (Sigma-Aldrich). NAT2 catalytic activity was quenched by addition of 99.8% methanol to each well after 30 min and compound separation was achieved by liquid chromatography (LC) using a C18 BEH column (1.7 µm, 2 × 50 mm; Waters) with mobile phases consisting of 10 mM ammonium carbonate, pH 10 (A) and acetonitrile (B). NAT2 substrate and product levels were detected through tandem mass spectrometry (MS/MS) in a XEVO TQ mass spectrometer (Waters), by monitoring the following m/z transitions: 284>239 (amonafide), 326>281 (N-acetyl-amonafide), 236>163 (procainamide), and 278>205 (N-acetyl-procainamide).
Procainamide
Manufactured by Waters Corporation
Procainamide is a laboratory equipment product manufactured by Waters Corporation. It is a chemical compound used in various analytical and research applications. Procainamide serves as a reference standard for the identification and quantification of procainamide in biological samples.
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Lab products found in correlation
Procainamide, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap mass spectrometry, by Thermo Fisher Scientific (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Procainamide, by Merck Group (1 mentions)
Xevo tq mass spectrometer, by Waters Corporation (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
2 protocols using procainamide
1
NAT2 Enzymatic Activity Assay
2
UPLC-Fluorescence Profiling of N-Glycans
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Known amount of N-glycans were tagged with fluorescent tag 2-aminobenzamide (2-AB) and/or procainamide (Sigma) and profiled using BEH-Amide column using Waters Acquity UPLC system equipped with online fluorescence detector. The excitation and emission wavelengths were set at 320 nm and 420 nm for 2AB and 310 nm and 370 nm for procainamide, respectively. We used a gradient mixture of 100 mM ammonium formate buffer at pH 4.5 and acetonitrile as running buffer for UPLC. The peaks eluted were compared with highly sialylated N-glycans isolated from bovine fetuin. We confirmed the structures by off-line mass spectral identification of the procainamide tagged N-glycans using LTQ-Orbitrap mass spectrometry (Thermo Scientific) in negative ionization mode. Briefly, selected N-glycan peaks were collected from UPLC, dried down by speed-vac, reconstituted in 50% aqueous methanol containing 10 mM ammonium formate (pH 4.5) and injected directly into ion-source from external syringe pump at a flow rate of 5μL/min. We confirmed by Mass Spectrometry the structural assignment given to each peak from UPLC-FL.
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