Reductive amination of the N‐glycans with Anthranilic acid (2‐AA, SigmaAldrich 10680) was performed to increase the MS signal and enable chromatographic separation on C18 columns. The labeling is highly efficient and the reduction of the sample also solves the problem of peak splitting of α and β anomers observed when analyzing native glycans. The labeling was performed as described by SigmaAldrich with some modifications. Briefly, a labeling solution with 60 mg/ml 2‐AA and 60 mg/ml sodium cyanoborohydride (SigmaAldrich 42077) in DMSO (SigmaAldrich 276855):acetic acid (70:30) was prepared. The solution was briefly heated in a heating block to 65°C and water was added to a final concentration of 10%. Labeling solution (5 µl) was added to the lyophilized N‐glycans and incubated at 65°C in a heating block under aluminum foil for 3 hr. Condensed liquid in the lid was briefly spun down a few times during the incubation. The reaction was stopped with the addition of 95 µl water and immediately applied to disposable size exclusion columns (SEC), see below.
Anthranilic acid 2 aa
Manufactured by Merck Group
Sourced in United States
Anthranilic acid (2‐AA) is a colorless crystalline solid compound with the chemical formula C6H7NO2. It is a derivative of benzoic acid and is used as an intermediate in the production of various pharmaceutical and agrochemical products.
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2 protocols using anthranilic acid 2 aa
1
Reductive Amination of N-Glycans with 2-AA
2
HPLC and MALDI-TOF Analysis of N-Glycans
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N-glycans were analyzed through HPLC and MALDI-TOF20 . Briefly, N-glycans were separated from 15 μg of MP by PNGase F (New England Biolabs, UK). After purification with a Carbograph solid-phase extraction column, the separated N-glycans were labeled with anthranilic acid (2-AA; Sigma-Aldrich, USA) and purified. Each dried N-glycan sample was dissolved in 50 μl of HPLC water and analyzed using normal-phase HPLC with a Shodex Asahipak NH2P-50 4E column (0.43 × 25 cm, SHOWA DENKO K.K., Japan), in 70% solvent A (1% tetrahydrofuran [THF] and 2% acetic acid in 100% Acetonitrile) and 30% solvent B (1% THF, 5% acetic acid, and 3% triethylamine in HPLC water), for 90 min and detected with a fluorescence detector at 360 and 425 nm wavelengths.
For MALDI-TOF analysis, N-glycans fractionated via HPLC were collected and dried. The matrix solution (the same ratio of 6-Aza-2-thiothymine solution and 2,5-Dihydroxy-benzoic acid solution) was mixed with the same volume of N-glycan sample. The sample was spotted on an MSP 96 polished-steel target (Bruker, Germany), and the crystallized sample was analyzed via MALDI-TOF (Bruker, Germany) in a linear negative mode.
For MALDI-TOF analysis, N-glycans fractionated via HPLC were collected and dried. The matrix solution (the same ratio of 6-Aza-2-thiothymine solution and 2,5-Dihydroxy-benzoic acid solution) was mixed with the same volume of N-glycan sample. The sample was spotted on an MSP 96 polished-steel target (Bruker, Germany), and the crystallized sample was analyzed via MALDI-TOF (Bruker, Germany) in a linear negative mode.
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