For N-glycosylation analysis, N-glycans were detached from mAbs via PNGase F (New England Biolabs, Ipswich, MA) and labeled by RapiFluor-MS (Waters, Millford, MA) according to the manufacturer’s protocol. N-glycans were analyzed using Acquity UPLC I-class system (Waters) with Acquity UPLC Glycan BEH amide column (Waters) according to the manufacturer’s protocol. The signal was monitored at 265 nm excitation and 425 nm emission wavelengths.
Rapifluor ms
Manufactured by Waters Corporation
Sourced in United States
RapiFluor-MS is a fluorescent labeling reagent designed for the sensitive detection of glycans in mass spectrometry analysis. It enables the rapid and efficient labeling of glycans, enhancing their detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pngase f, by New England Biolabs (2 mentions)
Acquity uplc glycan beh amide column, by Waters Corporation (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Rapigest sf, by Waters Corporation (1 mentions)
Glu c, by Promega (1 mentions)
Trypsin, by Promega (1 mentions)
Glycoworks hilic elution plate, by Waters Corporation (1 mentions)
Rapigest, by Waters Corporation (1 mentions)
Acquity h class uplc instrument, by Waters Corporation (1 mentions)
Recombinant pngase f, by New England Biolabs (1 mentions)
5 protocols using rapifluor ms
1
N-Glycan Analysis of Monoclonal Antibodies
2
Rapid N-Glycan Preparation and Labeling
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Sample denaturation, de-N-glycosylation, labelling with Rapifluor-MS (RFMS), and purification were performed in accordance with the Waters Corporation “GlycoWorks Rapifluor-MS N-Glycan Kit Care and Use Manual” (p/n 715004793) (Waters, Milford, MA, USA). Briefly, the glycoproteins were denatured at 90 °C for 3 min in the presence of 5% (w/v) RapiGest. De-N-glycosylation was then conducted at 50 °C for 5 min, with the addition of 1.2 μL of Rapid PNGase F. The digested samples were directly subjected to RFMS labelling without purification by adding 6 μL of RFMS reagent. The reaction was then allowed to proceed at room temperature for 5 min. A GlycoWorks μElution Plate was used for the SPE Clean-up procedure. Glycans were eluted with 200 mM ammonium acetate in 5% acetonitrile.
3
Characterization of Monoclonal Antibody Glycosylation
4
Rapid Protein N-Glycan Analysis
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Protein-A purified protein was denatured in 1% RapiGest TM (Waters) solution, followed by the simultaneous release of N-glycans using Rapid TM PNGaseF (New England BioLabs) and derivatization with RapiFluor-MS (Waters). The GlycoWorks™ SPE Reagents-Automation kit (Waters) and GlycoWorks™ HILIC µElution plate (Waters) were used to purify and concentrate the released and fluorescently labeled N-glycans.
5
IgG N-Glycan Preparation and Analysis
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All chemical reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). Human IgG was purchased from Sigma-Aldrich (I4506) for reduction, alkylation, and N-glycan labeling with 2-AB and AQC as sources for 2-AB-and AQClabeled IgG N-glycans. Waters RapiFluor-MS glycan performance standard (186007983) was used as a source of RapiFluor-MS-labeled IgG N-glycans. Recombinant PNGase F (P0709L), ABS (α2-3,6,8,9 Neuraminidase A, P0722L, 20 mU/mL), BTG (bovine testes β(1-3/4)-galactosidase, 2 mU/mL), GUH (β-N-Acetylglucosaminidase S, 4 mU/ml), and BKF (α1-2,3,4,6 Fucosidase, 2 mU/mL) were obtained from New England Biolabs (Ipswich, Massachusetts). BTG (PZGKX-5013, 5 U/mL), BKF (PZGKX-5006, 500 mU), and JBM (PZGKX-5010, 150 U/mL) were purchased from Prozyme (San Leandro, California). 10K Nanosep centrifugal devices were purchased from Pall (Port Washington, NY) and Hypersep Diol cartridges from Thermo Fischer Scientific (Waltham, Massachusetts). Samples were analyzed on a Waters Acquity H-Class UPLC instrument (Milford, MA).
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