PNGase F treatment of SPR-515 shows an observable shift in apparent MW, indicating the presence of N-glycosylation. Monomers were treated with PNGase F (NEB), a glycosidase which specifically cleaves between the innermost GlcNAc and asparagine residues of N-linked oligosaccharides, and incubated at 37oC for 1 hour and run on a 5-12% gradient Bis-Tris gel.

Modified/Total deamidation spectrum counts as determined by mass spectrometry of SPR-515 before and after PNGase F treatment identifies potential N-glycosylation sites at N167, N359 and N410. Blue color indicates deamidation sites that match the N-glycosylation motif (N-X-S/T/C) and have a higher deamidation count after PNGase F treatment. No deamidation was present at N167 or N410 without PNGase F, suggesting these residues are protected from nonspecific deamidation by N-glycosylation. Some deamidation was present at N359 without PNGase F treatment, indicating a population of monomers is not glycosylated at this position. Several non-consensus, non-PNGase F-dependent deamidation sites were present, which may have occurred during production or the mass spectrometry workflow. Both CID and HCD fragmentation methods were used to improve sequence coverage and deamidation detection. Overall protein sequence coverage was 82%, with a localization probability cutoff set at ≥95%.