The total glycan profiles of ExpiCHO and 293 F–produced trimers were generated by HILIC-UPLC. N-linked glycans were enzymatically released from Envs via in-gel digestion with peptide N-glycosidase F (PNGase F), subsequently fluorescently labeled with 2-aminobenzoic acid, and analyzed by HILIC-UPLC, as previously described (53, 54, 95, 96). Digestion of released glycans with Endo H enabled the quantitation of oligomannose-type glycans (95). The composition of the glycans was determined by analyzing released glycans from trimers by PNGase F digestion using ion mobility MS (53). Negative ion mass, collision-induced dissociation, and ion mobility spectra were recorded with a Waters SYNAPT G2-Si mass spectrometer (Waters Corp.) fitted with a nanoelectrospray ion source. Waters DriftScope (version 2.8) software and MassLynx (version 4.1) were used for data acquisition and processing. Spectra were interpreted as described previously (97100). The results obtained served as the basis for the creation of sample-specific glycan libraries, which were used for subsequent site-specific N-glycosylation analyses. For site-specific N-glycosylation analysis, before digestion, trimers were denatured and alkylated by incubation for 1 hour at room temperature in a 50 mM tris-HCl (pH 8.0) buffer containing 6 M urea and 5 mM dithiothreitol (DTT), followed by addition of 20 mM iodoacetamide (IAA) for a further 1 hour at room temperature in the dark and then additional DTT (20 mM) for another 1 hour to eliminate any residual IAA. The alkylated trimers were buffer-exchanged into 50 mM tris-HCl (pH 8.0) using Vivaspin columns and digested separately with trypsin and chymotrypsin (Mass Spectrometry Grade, Promega) at a ratio of 1:30 (w/w). Glycopeptides were selected from the protease-digested samples using the ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore). Enriched glycopeptides were analyzed by LC–electrospray ionization MS on an Orbitrap fusion mass spectrometer (Thermo Fisher Scientific), as previously described (53), using higher-energy collisional dissociation fragmentation. Data analysis and glycopeptide identification were performed using Byonic (version 2.7) and Byologic software (version 2.3; Protein Metrics Inc.), as previously described (53).

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