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.
Procainamide
Manufactured by Thermo Fisher Scientific
Sourced in United States
Procainamide is a pharmaceutical agent used as a class Ia antiarrhythmic drug. It functions by blocking sodium channels in the heart, which can help regulate abnormal heart rhythms.
Automatically generated - may contain errors
Lab products found in correlation
Procainamide, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Ltq orbitrap mass spectrometry, by Thermo Fisher Scientific (1 mentions)
Pngase f, by Promega (1 mentions)
Milli q water, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
α1 2 3 6 mannosidase, by Agilent Technologies (1 mentions)
Gly x instantpc labelling module, by Agilent Technologies (1 mentions)
Gly x instantpc cleanup module, by Agilent Technologies (1 mentions)
Gly x deglycosylation module gx96 100, by Agilent Technologies (1 mentions)
β1 4 galactosidase, by Agilent Technologies (1 mentions)
β 1 3 4 galactosidase, by Agilent Technologies (1 mentions)
β n acetylhexosaminidase, by Agilent Technologies (1 mentions)
3 protocols using procainamide
1
UPLC-Fluorescence Profiling of N-Glycans
2
IgG Glycan Preparation and Labeling
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Glycans from IgG protein were prepared according to the previously described protocol [44 (link)], which consists of three basic steps: glycan release from the protein, glycan labeling, and purification of glycans. Glycans were enzymatically released from IgG using PNGase F (Promega, Madison, WA, USA); the released IgG glycans were then labeled with a fluorescent dye procainamide (Thermo Fisher Scientific, Walthman, MA, USA) and purified using liquid chromatography solid-phase extraction (HILIC-SPE) on a 0.2 μm wwPTFE filter plate (Pall Corporation, Ann Arbor, MI, USA).
3
AdvanceBio Gly-X N-Glycan Preparation and Analysis
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The AdvanceBio Gly-X N-Glycan Prep with InstantPC kit, 96-ct (GX96-IPC) consisting of three modules, including Gly-X deglycosylation module (GX96-100), Gly-X InstantPC labelling module (GX96-101), and Gly-X InstantPC cleanup module (GX96-102), was donated by Agilent Technologies. The AdvanceBio Gly-X N-Glycan Prep with 2-AB express kit, 96-ct (GX96-2AB) consisting of three modules, including Gly-X deglycosylation module (GX96-100), Gly-X 2-AB express labelling module (GX96-401), and Gly-X 2-AB express cleanup module (GX96-402), were donated by Agilent Technologies. Advance-Bio exoglycosidases of Sialidase A (GK80040), Sialidase C (GK80030), β (1-3,4)-Galactosidase (bovine testis, GKX5013), β (1-4)-Galactosidase (Streptococcus pneumoniae), GKX5014), β-N-acetylhexosaminidase (GK80050), α (1-2,3,6)-Mannosidase (jack bean) (GKX-5010), and α
(1-2,3,4,6)-Fucosidase (bovine kidney) (GKX-5006) were donated by Agilent Technologies. Procainamide (10733211) was purchased from Thermo Fisher scientific. Human serum from human male AB plasma (H4522) was purchased from Sigma Aldrich. HPLC-grade acetonitrile was purchased from Sigma and Milli-Q water was used in all preparations. All the common chemicals were purchased from Sigma Aldrich.
(1-2,3,4,6)-Fucosidase (bovine kidney) (GKX-5006) were donated by Agilent Technologies. Procainamide (10733211) was purchased from Thermo Fisher scientific. Human serum from human male AB plasma (H4522) was purchased from Sigma Aldrich. HPLC-grade acetonitrile was purchased from Sigma and Milli-Q water was used in all preparations. All the common chemicals were purchased from Sigma Aldrich.
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