Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains 
multiple N- and O-glycosylation sites. An in-depth characterization of the 
glycosylation of etanercept was carried out using liquid chromatography/mass 
spectrometry (LC/MS) methods in a systematic approach in which we analyzed the 
N- and O-linked glycans and located the occupied O-glycosylation sites. 
Etanercept was first treated with peptide N-glycosidase F to release the 
N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) 
fluorescence tag and separated using ultraperformance liquid 
chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC). 
Preliminary structures were assigned using Glycobase. These assignments, which 
included monosaccharide sequence and linkage information, were confirmed by 
exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of 
the N-glycans from etanercept facilitated the selective characterization of 
O-glycopeptides and enabled the O-glycans to be identified. These were 
predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr 
residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on 
the O-glycans allowing the use of an automated LC/MS(E) protocol to identify the 
O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint 
the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and 
O-glycosylation sites in etanercept provides a basis for future studies 
addressing the biological importance of specific protein glycosylations in the 
production of safe and efficacious biotherapeutics.