A multi-ligand polymer-particle composite-based array chip for selective and simultaneous enrichment of...

In proteomic studies, efficient and selective enrichment of target phosphoproteins and glycoproteins from biological samples is of prime importance. An integrated phosphoprotein and glycoprotein enrichment platform could reduce several pre-and post-enrichment steps. In this study, we report the development of a multi-ligand polymer-particle composite integration within a single chip to simultaneously enrich phosphoproteins and glycoproteins from the same sample. TiO₂ particles (200-700 nm) were synthesized and characterized using FT-IR and FE-SEM. Two lectins, Concanavalin A (Con A) and peanut agglutinin (PNA), were individually immobilized on epoxide and aldehyde functionalized TiO₂ particles. FT-IR, XPS, and BCA assay have confirmed lectin immobilization. Of different TiO₂ particle surface modifications (bisoxirane, epichlorohydrin, silanization, and silanization followed by glutaraldehyde), lectins were immobilized with > 2-fold on epichlorohydrin and silane functionalized particles. The two glycoproteins (glucose oxidase (GOD) and ovalbumin (OVAL)) were captured with high adsorption by Con A immobilized particles (modified with glutaraldehyde linkers). Lectin functionalized particles and TiO₂ particles were individually incorporated within the PolyHEMA monolith structure (FE-SEM images) and fabricated as thin film spots (5 μL) on the glass slides. The maximum time required for the array chip to adsorb glycoproteins and phosphoproteins was 80 min and 120 min, respectively. Nano-LC-MS/MS analysis has confirmed the successful and selective enrichment of both model phosphoproteins (α-casein, and β-casein) and glycoproteins (GOD and OVAL) from an artificial protein mixture comprising of phosphoproteins, glycoproteins, non-phosphoproteins and non-glycoproteins.