Comparative assessment of glycosylation of recombinant human ...（七）

Figure 3 MS2 spectra of 2-AB-labeled N-glycan structures. Diagnostic ions are marked with corresponding fragment structures. (a) NeuGc1NeuAc1HexNAc4Hex5 from rhFSH [(M+2H)2+ at m/z 1180.34]. (b) NeuAc2HexNAc4Hex5SO41 from rhFSH, [(M+Na+H)2+ at m/z 1214.36]. *The NeuAc was dehydrated. (c) and (d) The two isomers of Fuc2NeuAc1HexNAc5Hex5 from uhFSH, [(M+2H)2+ at m/z 1274.48].

Figure 4 Site-specific characterization and comparison of N-glycosylation of rhFSH and uhFSH. (a) MS2 spectrum of the N-glycopeptide KVEN[+NeuAc2HexNAc4Hex5]HTAC#H,[(M+3H)3+ at m/z 1100.76]. The sequential losses of monosaccharide residues are assigned. (b) Comparative description of the glycans present at αN52 of rhFSH and uhFSH. The N-glycan structures were assigned based on previous reports.1, 32

Figure 5 Identification of the O-linked glycosylation sites in rhFSH and uhFSH. (a) HCD spectrum of the O-glycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of rhFSH. (b) ETD spectrum of the O-glycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of uhFSH. The O-glycosylation sites are both in red, boldface and italics. # represents alkylation of cysteine.

Figure 1 Glycoproteomic strategy and hFSH glycosylation characterization. (a) Strategy for the comparative assessment of glycosylation in rhFSH and uhFSH. (b) SDS-PAGE and MALDI-TOF mass spectra of both hFSHs. (c) IEF result (pH gradient: 3–6) of both hFSHs. (d) Sialic acid contents (n=2). Error bars represent SD from duplicate determinants. (e) Mass spectrum of non-glycosylation site at FC#ISINTTWC#AGYC#YTR [(M+3H)3+ at m/z 724.98] without the 0.98 Da increase after PNGase F digestion. # represents alkylation of cysteine.266x187mm (150 x 150 DPI)

Figure 5 Identification of the O-linked glycosylation sites in rhFSH and uhFSH. (a) HCD spectrum of the Oglycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of rhFSH. (b) ETD spectrum of the O-glycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of uhFSH. The Oglycosylation sites are both in red, boldface and italics. # represents alkylation of cysteine. 834x820mm (72 x 72 DPI)