PNGase F deaminates the asparagine residue to which the N-linked glycan sellekchem is attached and converts it to aspartic acid. If the glycan is of the high-mannose form, it will be sensitive to Endo H, which leaves one N-acetylglucosamine (GlcNAc) bound to the Asn. Thus, the gain of 1 Da (Asn to Asp; nitrogen to oxygen) by PNGase F or 203 Da by the GlcNAc residue left by Endo H digestion can be resolved by MS of the peptides. From the resulting three spectra (untreated, Endo H, and PNGase F treated), we are able to map all the glycosylation sites, estimate the approximate usage of each site, and determine whether the glycan at a particular site was complex or high mannose. We achieved almost 100% coverage of the eE2 sequence using either trypsin or chymotrypsin proteases.

Peptides from the 11 predicted N-linked glycosylation sites were shown to be fully glycosylated, since we were unable to detect unglycosylated peptides with Asn residues in them (Fig. (Fig.2B,2B, upper spectra of each panel). Only one of the 11 glycosylation sites was found to be Endo H sensitive (VGGVEHRLTAACNF; data not shown for Endo H), suggesting that the majority of the glycans are complex in nature (Fig. (Fig.2B,2B, lower spectra of each panel). Peptides containing the potential O-linked glycosylation sites were resolved and shown to be unmodified (data not shown). Oligomeric state and secondary structure of eE2. Since previous reports have shown that E2 tends to aggregate, we set out to define the oligomeric state of eE2, using nonreducing SDS-PAGE, size exclusion chromatography (SEC), and analytical ultracentrifugation.

SDS-PAGE analysis of eE2 under nonreducing conditions demonstrated that eE2 consisted of a mixture of two components with approximate molecular masses of ~120 kDa (dimer) and ~60 kDa (monomer), with a small amount of larger-molecular-mass protein (Fig. (Fig.3A).3A). To determine the oligomeric state of eE2 under native conditions, the protein was evaluated by SEC. eE2 yielded two major peaks and a slight peak found in the void volume of the column (Fig. (Fig.3B).3B). The major and minor peaks were measured at ~123 kDa and ~75 kDa, respectively, by an inline static light-scattering detector (data not shown). Both nonreducing SDS-PAGE and native SEC clearly demonstrated that eE2 is not aggregated. The ratio of dimer to monomer in nonreduced SDS-PAGE (Fig. (Fig.

3A)3A) and that in gel filtration (Fig. (Fig.3B)3B) are similar, suggesting that a portion of the dimer may result from an intermolecular disulfide bond. FIG. 3. (A) SDS-PAGE analysis of purified eE2 in the presence and absence of ��-mercaptoethanol Dacomitinib (��-ME) and stained with Coomassie blue. (B) Purified eE2 was applied to a Superdex 200 size exclusion column equilibrated with 50 mM HEPES (pH 7.5), … There are 18 conserved cysteines in full-length E2, which could result in the formation of 9 disulfide bonds. The eE2 fragment contains only 17 cysteines, leaving at least 1 unpaired.