Figure 3. Detection of specific fucosylated proteins.

(a) Slc35c1 transports GDP-fucose from the cytosol into the Golgi. Fut9 transfers fucose in α-1,3 linkage to LacNAc structures. Deletion of Slc35c1 abolishes fucosylation of secretory proteins in general. Ablation of Fut9 specifically affects synthesis of the Lewis X glyco-epitope. (b) Quantitative proteomics workflow for intact glycopeptides from Slc35c1 and Fut9 mutant (KO) and their genetically repaired (WT) mESC sister clones. (c) Quantitative glycoproteomics detects the reduction of all fucosylated glycopeptides (red) and the reciprocal increase of non-fucosylated glycopeptides (green) in Slc35c1 KO vs WT mESCs. Ablation of Fut9 affects a specific subset of fucosylated glycopeptides. Oligo-mannosidic type N-glycopeptides (blue) were not affected. Differential glycopeptide expression is shown by scatter-plotting TMT-reporter ion intensities for Slc35c1 KO (upper panel) and Fut9 KO (lower panel) versus WT cells. Data-points of Igf2r glycopeptide (ASIN¹⁵³²ASYSEK) glycoforms are indicated by circles. (d) Site-specific comparison of Igf2r glycopeptides. Differential glycopeptide expression is shown as the ratio of TMT-reporter ion intensities in Slc35c1 and Fut9 KO versus WT cells. The number of PSMs (n) considered for ratio calculations and box-plot representations, are indicated.