Magnetic boronate-Ti(IV) dual-affinity nanoparticles for simultaneous enrichment and sequential elution of glycopeptides and phosphopeptides.

The simultaneous enrichment of glycopeptides and phosphopeptides from complex peptide mixtures remains challenging because boronate affinity and immobilized metal ion affinity chromatography (IMAC) typically require different pH conditions for efficient binding. We evaluated the previously reported dual boronate-Ti(IV) magnetic nanomaterial, Mag@DBA-Ti(IV), as a sample-preparation platform for the single-sorbent, single-loading-condition enrichment and sequential elution of glycopeptides and phosphopeptides. The material, prepared by co-immobilizing a low-pKₐ boronic acid ligand and alendronate on magnetic nanoparticles, followed by Ti(IV) chelation, was originally developed for small-molecule enrichment; its translation to peptide-level proteomics is established here. The integrated boronate and Ti(IV)-IMAC sites enabled concurrent binding of glycopeptides and phosphopeptides under neutral loading conditions, with selective release achieved using acidic and alkaline eluents, respectively. This translation required redesigning the elution chemistry for MS compatibility, replacing the high-pH sodium phosphate eluent used for small molecules with 0.4 mol L⁻¹ NH₃·H₂O, and establishing that the dual-affinity sites retain selectivity when glycopeptides and phosphopeptides are low-abundance species competing with a large excess of unmodified peptides. Mag@DBA-Ti(IV) exhibited high adsorption capacities toward model protein digests, reaching 71.4 mg g⁻¹ for IgG digest and 125 mg g⁻¹ for α-casein digest. The material also showed high selectivity for both glycopeptides and phosphopeptides in the presence of a 500-fold excess of bovine serum albumin digest, allowing detection at concentrations as low as 10⁻¹⁰ mol L⁻¹. When applied to U251 cell lysate digests, Mag@DBA-Ti(IV) enabled the identification of 1,945 glycopeptides from 752 glycoproteins and 7,498 phosphopeptides from 2,410 phosphoproteins in a single experiment. These results demonstrate that the dual boronate-Ti(IV) affinity concept, originally established for small molecules, can be translated to peptide-level bottom-up proteomics, providing a single-sorbent route to integrated glycoproteomic and phosphoproteomic determination.