The samples were immobilized in gels and N-glycans were removed from all samples using PNGase F [20 (link)]. O-glycans were released using microwave-assisted non-reductive β-elimination and labelled with 2-aminobenzamide (2AB) (75%) and with reductive β-elimination (25%) as described in Wilkinson et al [21 (link)].
Microwave-assisted non-reductive β-elimination was performed as described in Wilkinson et al. [21 (link)]. Briefly, the gels containing O-glycans were crushed, lyophilized, and resuspended in 250 μL 40% dimethylamine in water with 0.1 g/mL ammonium carbonate. Samples were incubated in a Monowave 450 microwave reactor at 600W (Anton Paar) under 12-minute microwave radiation at 70°C. Samples were then neutralized with 1M HCl, desalted using a Hypersep Hypercarb SPE cartridge, lyophilized and fluorescently labelled with 2AB [22 (link)]. The label excess was removed by a Hypersep Diol SPE cartridge [23 (link)].
Reductive β-elimination and desalting was performed according to Schulz et al [24 (link)] and modified for an in-gel release [25 (link)].
Monowave 450 microwave reactor
Manufactured by Anton Paar
The Monowave 450 microwave reactor is a laboratory instrument designed for high-throughput chemical synthesis and sample preparation. The device utilizes microwave technology to provide efficient and reproducible heating for a wide range of chemical reactions and applications.
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Lab products found in correlation
3 protocols using monowave 450 microwave reactor
1
Efficient Glycan Release and Labeling
2
N-Glycan Microwave-Assisted Release
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Gels from N-glycan release were crushed by passing them through a small hole
in the bottom of a 0.2 mL tube into a 1.5 mL tube (under 14,000 rpm
centrifugal force), dried down, resuspended in 250 μL 40% dimethylamine
in water containing 0.1 g/mL ammonium carbonate, and transferred to
G4 vials (Anton Paar). Tubes were subjected to 12 min microwave radiation
at 70 °C in a Monowave 450 microwave reactor at 600 W (Anton
Paar). The reaction solution, including gel, was transferred to 2
mL tubes and neutralized with 1 M HCl. Samples were desalted using
a HyperSep Hypercarb SPE cartridge and dried.
in the bottom of a 0.2 mL tube into a 1.5 mL tube (under 14,000 rpm
centrifugal force), dried down, resuspended in 250 μL 40% dimethylamine
in water containing 0.1 g/mL ammonium carbonate, and transferred to
G4 vials (Anton Paar). Tubes were subjected to 12 min microwave radiation
at 70 °C in a Monowave 450 microwave reactor at 600 W (Anton
Paar). The reaction solution, including gel, was transferred to 2
mL tubes and neutralized with 1 M HCl. Samples were desalted using
a HyperSep Hypercarb SPE cartridge and dried.
3
Extraction and Characterization of O-Glycans
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Our group has recently developed a protocol for the extraction and characterization of O-linked glycans (Wilkinson et al. 2021 (link)) with the following modifications. The remaining gels after N-glycan release were crushed by passing them through a small hole in the bottom of a 0.5 mL tube into a 1.5 mL tube at 14,000 rpm and dried down. Then, the crushed gels were resuspended in 250 μL of 40% dimethylamine in water containing 0.1 g/mL ammonium carbonate and transferred to G4 vials (Anton Paar, Graz, Austria). The vials were inserted into a Monowave 450 microwave reactor (Anton Paar), in which the vials were subjected to 20 min microwave radiation at 70°C and 600 W. After the reaction, the remaining solution including the pieces of gel were transferred to 2 mL tubes and neutralized with 1 M HCl (all samples were at pH 7). Samples were desalted using a Hypersep Hypercarb SPE cartridge (Thermo Fisher Scientific, Waltham, MA) and dried.
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