Plasma membrane pellets were resuspended with 100 μL of 5 mM DTT in 100 mM NH4HCO3 and heated in a 100 °C water bath for 1 min to thermally denature the proteins. The cleavage of N-glycans was performed by adding 2 μL of peptide N-glycosidase F (PNGase F, New England Biolabs, MA) followed by the incubation at 37 °C in a microwave reactor (CEM Corporation, NC) for 10 min at 20 watts. To convert the N-glycans from amines to aldehydes, the sample was allowed to stand in a 37 °C water bath overnight. To separate the released N-glycans from the membrane fractions, residual deglycosylated proteins and lipids, 350 μL of nanopure water was added and ultra-centrifugation at 200 000 × g was conducted for 45 min. The released N-glycans contained in the supernatant were purified using porous graphitic carbon (PGC) on an SPE plate (Grace, IL) and eluted with 40% (v/v) ACN and 0.5% (v/v) TFA in water. The eluted samples were dried in vacuo and stored at –20 °C until analysis.
Spe plate
Manufactured by Grace Bio-Labs
Sourced in United States
The SPE plate is a solid-phase extraction (SPE) device designed for sample preparation. It facilitates the selective separation and concentration of target analytes from complex matrices. The SPE plate functions by allowing the sample to pass through a sorbent material, which retains the analytes of interest while allowing interfering components to be washed away.
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2 protocols using spe plate
1
N-Glycan Release and Purification
2
Glycomic Sample Preparation and N-Glycan Isolation
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Details of the glycomic sample preparation have been described previously ((Wu et al., 2010 (link); Wu et al., 2011 (link))). Extracted cell membrane fractions or protein (RNase B) were suspended with 100 μl of 100 mM NH4HCO3 in 5 mM dithiothreitol and heated in boiling water for 2 min to denature the proteins. Solutions of with 2 μl of peptide N-glycosidase F (New England Biolabs, MA, United States) were added to the samples, and the resulting solutions were then incubated in a microwave reactor (CEM Corporation, NC, United States) at 20 W, 37°C for 10 min. The samples were further placed in a 37°C water bath for 18 h. Ultracentrifugation at 200,000 × g for 45 min was performed to precipitate proteins, and the supernatant containing N-glycans was collected and desalted using porous graphitic carbon (PGC) on a 96-well SPE plate (Grace, IL, United States). The plate was equilibrated with 80% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Then the samples were loaded onto the plate and washed with nanopure water. N-Glycans were eluted with a solution of 40% (v/v) acetonitrile containing 0.05% (v/v) trifluoroacetic acid, and dried in vacuo using miVac (SP Scientific, PA, United States) prior to further analysis.
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