Interface-Engineered Hollow Nanospheres with Titanium(IV) Binding Sites and Microwindows as Affinity Probes for Ultrafast and Enhanced Phosphopeptides Enrichment

Enrichment and identification of phosphopeptides in real biological samples are of great significance in many aspects. Herein, Ti⁴⁺-immobilized silica hollow nanospheres were tailored via chelating with phosphonic acid groups produced from dealkylation of phosphonate ester functionalized silica hollow nanospheres, which were synthesized through a single micelle templated method with diethylphosphatoethyltriethoxysilane (DPTES) and tetramethoxysilane (TMOS) as silane precursors under neutral conditions. The characterization results of transmission electron microscopy (TEM), nitrogen sorption isotherms, FT-IR, and energy-dispersive X-ray (EDX) spectroscopy confirmed the successful preparation of Ti⁴⁺-immobilized silica hollow nanospheres (SHS-Ti; approximately 17 nm particle size), which possessed a 10 nm hollow cavity with 1.6 nm micropores on the thin shell (about 3.5 nm). Attributed to the immobilized Ti⁴⁺ and high specific area (396 m²/g), SHS-Ti was applied as a Ti⁴⁺-immobilized metal affinity chromatography (Ti-IMAC) material and showed good specificity, a low limit of detection (5 fmol), high selectivity (tryptic digestion mixture of bovine serum albumin/β-casein, 1000:1 molar ratio), high binding capacity (120 mg/g for pyridoxal 5′-phosphate), and a high binding constant (1.30 × 10³ L/mg). Particularly, benefiting from the unique hollow structure with microwindows on the thin shell, a short transport path, and small mass transfer resistance, SHS-Ti exhibited excellent enrichment speed in which both phosphopeptide loading and elution could be completed in 1 min. The 5298 unique phosphopeptides from 1618 unique phosphoproteins were identified after enrichment by SHS-Ti from 100 μg Jurkat cell lysates within three independent replicates. The results showed that SHS-Ti could be utilized as a novel and promising enrichment probe for phosphopeptide characterization in MS-based phosphoproteomics and related fields.