Removal of N-Linked Glycans from Glycoproteins, Microwave deglycosylation, Proteomics, Glycomics…
Denaturing deglycosylation of 100 µg of glycoproteins and analysis
- Add 100 µg of glycoprotein and fill with UltraPure H2O (milliQ, NanoPure, etc) until 70 µl.
- Add 10 µl of Denaturing Buffer (5% SDS, 400 mM DTT).
- Incubate at 100°C for 10 minutes.
- Add 10 µl of reaction buffer (500 mM Sodium Phosphate (pH 7.5)) and 10 µl of 10% NP-40.
- Add 0.1 µl of Carboclip PNGase F
- Incubate at 37°C for 30 min.
(Depending on the glycoprotein up to overnight incubation could be necessary)
- Analyze by SDS-PAGE
Non-denaturing deglycosylation of IgG:MALDI-TOF compatible application.
1. IgG desalting using an Amicon Ultra-0.5 mL Centrifugal Filter device MWCO 30 kDa:
Add your IgG sample to the filter device. If volume is less than 400 µL, add ultra pure water up to 400 µL and spin the sample for 5 minutes at 9,000x g at room temperature. Discard the flow-through. Repeat for a total of three times.
Desalting of antibody is recommended as the presence of salts and detergents could affect the MALDI-TOF-MS acquisition.
2. Antibody Deglycosylation:
Add 3-6 µL of reconstituted CarboClip enzyme for each 50-200 µg of IgG to deglycosylate. Mix the sample by flicking (do not vortex or pipette up and down). Incubate the mixture at 60°C for 1 hour with gentle shaking.
For efficient deglycosylation of the Fab region IgG denaturation could be necessary.
Analysis of protein deglycosylation by MALDI-TOF.
e.g. Deglycosylation of IgG under non-denaturing conditions
- Add 2 µl of PNGase F (CarboClip) to 100 µl of IgG (10 µg/µl) diluted in nanoPure H2O.
- Incubate at 60 ºC for 2h.
- Clean and concentrate the sample using an amicon device
- Add 1 µl of concentrated IgG to the polished steel MALDI-TOF plate,
- Add 1 µl of SuperDHB (diluted in ACN at 20 µg/µl).
- Let it dry and analyze by MALDI-TOF
- Flex control-ultraflex tof-tof software
Set laser Attenuator “3_medium”.
Mass range 68,000 – 170,000.
See full protocol: IgG deglycosylation analysis using MALDI-TOF
Parsimonious Charge Deconvolution for Native Mass Spectrometry. Journal of Proteome Research. 2018
Marshall Bern, Tomislav Caval, Yong J. Kil, Wilfred Tang, Christopher Becker, Eric Carlson, Doron Kletter, K. Ilker Sen, Nicolas Galy, Dominique Hagemans, Vojtech Franc, and Albert J. R. Heck*
Marton Szigeti , Jeff Chapman , Beata Borza and Andras Guttman*