The enzymatic release and RFMS labelling of N-glycans was performed according to the Waters Glycoworks RapiFluor-MS N-Glycan Kit Care and Use Manual [28 ]. The protocol was carried out by introducing a glycoprotein quantity of 15 µg for each antibody product. The obtained RFMS derivatized N-glycans were directly analysed after labelling. For the RFMS label, approximately 5 pmol of labelled glycan material was loaded onto the column.
Glycoworks rapifluor ms n glycan kit
Manufactured by Waters Corporation
Sourced in United States
The GlycoWorks RapiFluor-MS N-Glycan Kit is a laboratory product that enables the fluorescent labeling of N-glycans derived from glycoproteins for analysis using mass spectrometry. The kit provides the necessary reagents and protocols to facilitate the sample preparation and labeling process.
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Lab products found in correlation
Thermo xcalibur software, by Thermo Fisher Scientific (6 mentions)
Amicon ultra 15, by Merck Group (3 mentions)
Velos pro iontrap ms, by Thermo Fisher Scientific (2 mentions)
Minute plasma membrane protein isolation kit, by Invent Biotechnologies (1 mentions)
Acetonitrile of lc ms grade, by Merck Group (1 mentions)
Agilent 1260 infinity 2 hplc system, by Agilent Technologies (1 mentions)
Ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
Masshunter qualitative analysis software package, by Agilent Technologies (1 mentions)
Agilent 1200sl hplc system, by Agilent Technologies (1 mentions)
Acquity uplc flr detector, by Waters Corporation (1 mentions)
Xevo g2 xs qtof, by Waters Corporation (1 mentions)
Nanoacquity, by Waters Corporation (1 mentions)
19 protocols using glycoworks rapifluor ms n glycan kit
1
Enzymatic Release and RFMS Labeling of N-Glycans
2
IgG N-Glycan Analysis by RapiFluor-MS
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Deglycosylation, RapiFluor-MS labelling and purification of IgG N-glycans were performed using the GlycoWorks RapiFluor-MS N-Glycan Kit obtained from Waters Corporation (USA). The entire procedure was done following Waters’ protocol (Waters Corporation, 2017, https://www.waters.com/webassets/cms/ support/docs/715004793en.pdf), which was further adapted to suit the high-throughput analysis in the 96-well PCR plate format (Deriš et al., 2021 (link)). Isolated and dried IgG was first resuspended in 10.8 μL ultrapure water, swiftly denatured with 3 μL 5 % RapiGest SF solution in a 3 min reaction at 99°C and enzymatically deglycosylated with 1.2 μL GlycoWorks Rapid PNGase F in a 5 min reaction at 50°C. Released N-glycans were then labeled with the RapiFluor-MS label. RapiFluor-MS dye contains a rapid tagging function group, N-hydroxysuccinimide carbamate, which rapidly modifies glycosylamine-bearing N-glycans after their enzymatic release, yielding a stable urea linkage (Lauber et al., 2015 (link)). Labeled N-glycans were purified by hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE) clean-up. In the end, the samples of released and labelled IgG glycans were stored at -20 °C until further use.
3
N-Glycan Profiling of Plasma Membrane
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Glycoworks RapiFluor-MS N-Glycan kit was purchased from Waters Corporation (MA, USA). Minute™ plasma membrane protein isolation kit was purchased from Invent Biotechnologies (MN, USA). IgG from porcine serum, fetuin from fetal bovine serum, lactoferrin from bovine milk and ribonuclease B from bovine pancreas were all purchased from Sigma-Aldich (St. Louis, MO USA). Acetonitrile of LC–MS grade was purchased from Merck (Darmstadt, Germany). Ultra-pure water was prepared by ELGA LabWater (resistivity ≥18.2 MΩ×cm, 25 °C).
4
N-Glycan Oligosaccharide Analysis by HPLC-MS
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N-Glycan oligosaccharide analysis was performed30 using the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters Corporation, Milford, MA). Fluor-MS N-glycan analysis was performed using an Agilent 1260 Infinity II HPLC system equipped with a 1260 FLD detector and an Agilent 6230 electrospray ionization Time-of-Flight mass spectrometer (Agilent, Santa Clara, CA). A HILIC AdvanceBio Glycan Mapping column (120 Å, 2.1 × 150 mm, 2.7 μm), operated at 45°C, was used to separate various N-glycans. Fluorescence was obtained using excitation and emission wavelengths of 265 and 425 nm, respectively. MS was acquired simultaneously from 400 to 2000 m/z at a constant scan rate of one spectrum per second. N-glycans were assigned based on m/z values using a N-glycan database (Water/NIBRT Glycan 3+) and N-glycan quantification was calculated on integration of the fluorescence chromatogram.
Site-specific glycosylation analysis was also performed as described previously.52 (link)
Site-specific glycosylation analysis was also performed as described previously.52 (link)
5
IgG N-Glycan Analysis by RapiFluor-MS
6
IgG Isolation and N-Glycan Analysis
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IgG was isolated using a 96-well protein G monolithic plate (BIA Separations, Slovenia, Cat No. 120.1012-2)19 using a protocol described by Trbojević-Akmačić and colleagues.20 (link) After IgG isolation, IgG eluates were heated at 65°C for 30 minutes to reduce the risk of any potential residual virus in the IgG eluate. An appropriate volume of IgG was aliquoted in a PCR plate (Thermo Scientific, UK, Cat. No. AB1300) and dried in a vacuum centrifuge, if needed. Deglycosylation, released N-glycan labeling and clean-up were performed using two different kits, GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, USA, Cat. No. 176003910) and AdvanceBio Gly-X N-glycan Prep with InstantPC Kit (Agilent, USA, Cat. No. GX96-IPC) according to the manufacturer's instructions.
7
N-Glycan Profiling of Secretome Proteins
8
N-Glycan Analysis of Secretome
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Example 3
N-glycan analysis was performed with GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, Mass.) according to the manufacturer's instruction. In this case 12 μl of 10× concentrated (MWCO filtered, Amicon Ultra-15, Merck, Darmstadt, Germany) secretome or purified protein sample were used for each. Labeled N-Glycans were analyzed by a LC-MS system using a Thermo Ultimate 3000 HPLC with fluorescence detector coupled on-line to a Thermo Velos Pro Iontrap MS. Separation gradient 30% to 43% buffer and MS was run in positive mode.
9
N-Glycan Profiling of Therapeutic Proteins
10
Automated N-glycan Release and Labeling for Mass Spectrometry
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The N-glycans were prepared using the GlycoWorks RapiFluor-MS N-glycan kit (Waters) on an
Andrew Alliance pipetting robot (Geneva, Switzerland) as described previously.19 (link) Briefly, 10 µL of diluted infliximab sample was loaded to a 96-well sample plate.
Reagents including RapiGest surfactant, Rapid PNGase F enzyme, and RFMS
labeling reagent were used for protein denaturation, N-glycan release, and labeling,
respectively, by following a modified protocol in the GlycoWorks RapiFluor-MS
N-Glycan Kit Care and Use Manual (p/n 715004793).19 (link) A total of 48 released N-glycan samples were prepared, including 12 samples from
batch 1 of innovator infliximab, 6 samples from each of the other three batches of
innovator infliximab, 6 samples for each of two batches of biosimilar infliximab, and 6
samples from a blank control solution. After the glycan labeling protocol was completed,
0.25 pmol of a high mannose glycan standard (Waters) was spiked into each of six released
glycan samples prepared from innovator batch 1. This group of samples was used to evaluate
the capability of the workflow to capture the relative abundance changes in glycans across
samples.
Andrew Alliance pipetting robot (Geneva, Switzerland) as described previously.19 (link) Briefly, 10 µL of diluted infliximab sample was loaded to a 96-well sample plate.
Reagents including RapiGest surfactant, Rapid PNGase F enzyme, and RFMS
labeling reagent were used for protein denaturation, N-glycan release, and labeling,
respectively, by following a modified protocol in the GlycoWorks RapiFluor-MS
N-Glycan Kit Care and Use Manual (p/n 715004793).19 (link) A total of 48 released N-glycan samples were prepared, including 12 samples from
batch 1 of innovator infliximab, 6 samples from each of the other three batches of
innovator infliximab, 6 samples for each of two batches of biosimilar infliximab, and 6
samples from a blank control solution. After the glycan labeling protocol was completed,
0.25 pmol of a high mannose glycan standard (Waters) was spiked into each of six released
glycan samples prepared from innovator batch 1. This group of samples was used to evaluate
the capability of the workflow to capture the relative abundance changes in glycans across
samples.
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