Beckman Coulter PA 800 plus - Enzymatic Release of the N-Linked Oligosaccharides; Working with the Supernatant; Principle of the Labeling Method

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Carbohydrate Labeling and Analysis Kit
Releasing Oligosaccharides From Glycoproteins
Enzymatic Release of the N-Linked Oligosaccharides
1. Aliquot the sample volume in order to have between 25-300 μg of glycoprotein.
2. Dry the glycoprotein solution completely in a centrifugal vacuum evaporator.
3. Add 45 μL of 1X PBS buffer.
4. Add 1.0 μL of 5% SDS (or a volume that gives final concentration of 0.1% SDS).
5. Add 1.5 μL of 1:10 dilution of 2-mercaptoethanol in DDI water (or a volume that gives final
concentration of 50 mM 2-mercaptoethanol).
6. Heat the sample for 5 minutes at 100°C.
IMPORTANT
If the denatured protein precipitates, discard the sample and restart this process, repeating
steps 1 through 5, and for step 6, denature the protein by incubating it at 37°C for 10 minutes.
7. Cool the sample to room temperature.
8. Add 5 μL of Nonidet® NP40 non-ionic detergent (or a sufficient volume that gives a final
concentration of 0.75%).
9. Add PNGase F enzyme, according to its activity.
10. React for 15 hours at 37°C.
11. Add approximately 150 μL of cold ethanol (or 3 times the actual reaction mixture volume).
12. Vortex the mixture and place it on ice for complete protein precipitation.
13. Centrifuge the samples at 15,000 rpm for 5 minutes.
14. Withdraw and save the supernatant.
IMPORTANT
This solution contains the RELEASED N-LINKED OLIGOSACCHARIDES.
15. Discard the solid. This is the deglycosylated protein.
Working with the Supernatant
There are two options for working with the supernatant:
For quantitative analysis, add an internal standard to the supernatant. See Preparing the Standards
beginning with Step 1a.
For qualitative analysis, bring the supernatant to dryness in the centrifugal vacuum evaporator.
These oligosaccharides are ready to be labeled with APTS. See Performing the Labeling Reaction in
this chapter.
Principle of the Labeling Method
After release (enzymatic or chemical), the oligosaccharides can be labeled with a fluorophore called
8-aminopyrene-1,3,6=, 6-trisulfonate. The stoichiometry of labeling reaction is one APTS molecule
per molecule of oligosaccharide. Figure 1 illustrates the labeling reaction of an N-linked
oligosaccharide with APTS.