Preparation of a Magnetic Ti-IMAC Material Based on Thiol-Ene Click Reaction and the Application in Intact Phosphoprotein Enrichment

Protein phosphorylation is a crucial post-translational modification that regulates protein activity, cellular signaling, transcriptional regulation, and cell cycle control. However, the analysis of phosphoproteins in biological samples is often compromised by complex sample matrices and interference from high-abundance proteins. While the top-down phosphoproteomics strategy enables comprehensive analysis of post-translational modifications based on intact proteins, its requirement for higher protein purity due to low protein ionization efficiency poses stern challenges. Consequently, developing appropriate enrichment methods for phosphoproteins in practical samples becomes essential. Immobilized metal ion affinity chromatography (IMAC) represents a common strategy for phosphorylated protein separation and enrichment. Among metal ions, Ti4+ has gained widespread application as IMAC chelating ligands due to its capacity to form multiple coordination networks and its high selectivity for phosphorylated protein enrichment, leveraging the strong chelating ability of phosphate groups toward metal ions. This paper presents the design and preparation of a novel magnetic Ti-IMAC nanocomposite, MNP@MPTMS–VPA–Ti(IV). The material is modified with phosphate groups via facile thiol-ene click chemistry and then immobilizes Ti4+, enabling selective enrichment of intact phosphoproteins through IMAC affinity. The efficiency of enrichment was evaluated using subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and analysis. This Ti-IMAC material-based magnetic solid-phase extraction (MSPE)-MALDI-TOF MS protocol has been successfully applied to enrich intact phosphoproteins in milk and eel mucus with high selectivity, sensitivity, and suitability.