Extended Data Fig. 6: Top-down and bottom-up glycoproteomics characterizing Fc-MFSD6(L3) glycoforms.

(a) Protein sequence table showing all identified N- and O-glycosites found for the Fc-MFSD6(L3) construct. (b) Top-down MS analysis of Fc-MFSD6(L3) construct expressed from wild-type cells (HEK293FT) (bottom) and cells deficient in O-linked glycosylation machinery (HEK293T ΔGALE/ΔGALK2 knockout) (top). Heterogeneity, imparted by the various O-glycosylation sites and structures, is observed in the wild-type Fc-MFSD6(L3). These O-glycan modifications are ablated in the Fc-MFSD6(L3) produced in ΔGALE/ΔGALK2 cells, resulting in a substantial mass loss and simplification of the intact protein mass spectra, due to reduction of multiple overlapping ion signals from the various glycoforms. (c) Western blot of Fc-MFSD6(L3) produced in wild-type and O-linked glycosylation deficient cells. The proteins were incubated with StcE, a protease that specifically cleaves mucin-domain glycoproteins. (d) Tandem MS analysis by HCD confirming the presence of core fucosylated N-glycan structure (Man3GlcNAc2Fuc1) located on N207 (representative glycopeptide LnVSDTVTLPTAPNMNSEPTLQPQTGEITNR) and resolved by the cryo-EM map shown in Fig. 4b. All b and y fragment ions are represented by the cyan and pink fragment labels, respectively. (e) Representative tandem MS spectra showing the diversity of the various N-glycan structures (Man3GlcNAc5Fuc1, Man3GlcNAc6Fuc1, and Man3GlcNAc4Gal1NeuAc1Fuc1) characterized by HCD at N207 and represented in Fig. 4c. All b and y fragment ions are represented by the cyan and pink fragment labels, respectively. All spectral assignments are within 2 ppm of total mass error.