Method Article
Oxidative release of glycans from carbohydrate-conjugated vaccine
https://doi.org/10.21203/rs.3.pex-2153/v1
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This protocol provides a oxidative method to release glycans from glycoconjugates which was conjugated using p-nitrophenyl ester homobifunctional linker. The advantages of this protocol are its low costs and easy way to purified glycans from complicated samples, such as cell lysate, for glycomic analyses by LC-MS/MS.
Although the processing of carrier protein of the carbohydrate-conjugated vaccine in antigen presenting cells has been well studied, the knowledge of how the glycan part is processed is limited. To analyze the processed glycan oxidative releasing method was used.
Oxidative releasing method using household bleach has been used to release all types of free reducing N-glycans and O-glycan-acids from glycoproteins, and glycan nitriles from glycosphingolipids1. Here, we use oxidative method to release glycans from glycocojugate Globo H-CRM197 which was originally conjugated Globo H using p-nitrophenyl ester homobifunctional linker onto carrier protein CRM1972. After further clean up using C18 cartridge, the released glycans are directly useful in glycomic analyses by LC-MS/MS.
NaClO (Honeywell)
Acetonitrile (Sigma-Aldrich)
Formic acid (Sigma-Aldrich)
Sep-Pak C18 1 cc Vac Cartridge, 100 mg Sorbent per Cartridge (Waters, WAT023590)
Centrifuge
SpeedVac evaporator
1. Add NaClO to less than 100 μg glycoconjugates or fractions of glycoconjugate treated cell lysate in 100 μl PBS or appropriated lysis buffer to give a final concentration at 1%.
2. Shake the mixture at room temperature for 1 min.
3. Add 10 μl volume of 10% formic acid to the mixture to quench the reaction.
4. Centrifugate the mixture at for 5 min at 16,000´g at 4 ℃.
5. Activate a Sep-Pak C18 100 mg sorbent cartridge with 1 ml 50% acetonitrile/0.1% formic acid and then equilibrium the cartridge with 1 ml 0.1% formic acid twice.
6. Pass the supernatant in step 4 through the Sep-Pak C18 cartridge and wash the cartridge with 3 ml 0.1% formic acid three times.
7. Eluted the glycans with 1 ml 20% acetonitrile and 0.1% formic acid
8. Dry the eluate by SpeedVac evaporator. The glycans are ready to analyze by LC-MS/MS.
Oxidative release: 15 minutes
C18 clean up: 30 minutes
Evaporation: 12 to 24 hours
1. Song, X. et al. Oxidative release of natural glycans for functional glycomics. Nature methods 13, 528-534 (2016).
2. Huang, Y.L. et al. Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer. Proc Natl Acad Sci U S A 110, 2517-22 (2013).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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