Titanium particles incorporated polymer monolith microcolumn for phosphoprotein enrichment from biological samples

In proteomic studies, selective enrichment of target phosphoproteins from biological samples is of importance. Of various enrichment methods, affinity chromatography is widely preferred method. Development of micro-affinity columns with simple strategies are in constant demand. Here in this report, for the first time, we have embedded TiO₂ particles within the monolith structure in a single step. Fourier transform infrared spectroscopy and scanning electron microscope analysis has confirmed the successful incorporation of TiO₂ particles within the polymer monolith. Incorporation of 3-(trimethoxy silyl) propyl methacrylate within the poly(hydroxyethyl methacrylate) based monolith composition has enhanced its rigidity and one fold phosphoprotein (α-casein) adsorption capacity. Presence of only 66.6 µg of TiO₂ particles within the monolith has displayed a four-fold higher affinity to α-casein over the non-phosphoprotein i.e. bovine serum albumin. Under optimized conditions (TiO₂ particle and acrylate silane), the affinity monolith has a maximum adsorption capacity of ∼ 72 mg per gram monolith. Translation of TiO₂ particles-monolith into a microcolumn of 3 cm long and 19 µL volume was successful. α-casein was selectively separated from an artificial protein mixture of α-casein and BSA, α-casein spiked human plasma, and cow milk within 7 min.