The protein was very well expressed from the HEK cells as predicted and could be easily purified using a combination of Ni affinity and size exclusion chromatography. The fact that HEK cells grow in a protein free media also made purification much more simple. What was clear from the SDS-PAGE analysis was that the protein had a number of different glycosylated forms with 1, 2, 3 and 4 of the N-linked sites being glycosylated. Our approach to removing the problematic sugars was to grow the cells in the presence of the mannosidase inhibitor, kifunensine, which results in an oligosaccharide structure that is sensitive to endoglycosidase H (EndoH) cleavage of the oligosaccharide (Chang, VT et al., 2007). EndoH treatment of the protein leaves a single N-acetyl glucosamine with a mass of 203Da. When the protein was expressed in the presence of kifunensine, purified and treated with EndoH this resulted in deglycosylation as shown in the SDS-PAGE gel below.
The protein was then analysed using protein mass spectrometry to calculate the intact mass. This revealed a number of species that had a mass +203Da, +406Da, +609Da and +812Da corresponding to N-acetylglucosamine present at 1, 2, 3 or 4 sites on the protein, exactly as expected from the EndoH cleavage of the N-linked structures.
Intact mass was performed on the Sciex X500B system coupled to a Sciex Exion LC