Superhydrophilic Multichannel TiO2 Fibers for Simultaneous Enrichment of O-GalNAc Glycopeptides and Phosphopeptides

Protein O-glycosylation and phosphorylation, both of which occur on serine and threonine residues of the protein, regulate critical biological functions in a crosstalk manner. However, due to the microheterogeneity, low abundance, and tedious enrichment processes of O-GalNAc glycopeptides, current research on their crosstalk is severely hindered. Here, we synthesized multichannel TiO2 fibers with superhydrophilicity and consecutive inner channels by electrospinning, enabling simultaneous and efficient enrichment of O-GalNAc glycopeptides and phosphopeptides. A total of 183 O-GalNAc glycopeptides and 3198 phosphopeptides were enriched from the HT29 cell line with four-channel TiO2 fibers, which are 1.3- and 1.5-fold those enriched by the commercial TiO2 porous beads. The subsequent application of these materials to trastuzumab-resistant breast cancer cell lines and their counterparts led to the identification of 73 significantly altered O-GalNAc glycoproteins and 740 phosphosites, shedding light on the potential mechanisms underlying drug resistance. These findings underscore the intricate interplay between GalNAc-type O-glycosylation and phosphorylation in trastuzumab resistance mechanisms in breast cancer cells.