Phosphorylation is a protein post-translational modification (PTM) that plays an important role in cell signaling, cell differentiation, and metabolism. The hyper phosphorylated forms of certain proteins have been appointed as biomarkers for neurodegenerative diseases, and phosphorylationrelated mutations are important for detecting cancer pathways. Due to the low abundance of phosphorylated proteins in biological fluids, sample enrichment is beneficial prior to detection.Thus, a need to find new strategies for enriching phosphopeptides has emerged. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials manufactured to exhibit affinity for a target molecule. In this study, MIPs have been synthesized using a new approach based on the use of fumed silica as sacrificial support acting as solid porogen with the template (phosphotyrosine) immobilized on its surface. Phosphotyrosine MIPs were tested against a mixture of peptides and phosphopeptides by performing micro-solid phase extraction using MIPs (μMISPE) packed in a pipette tip. First, the capability of the materials to preferentially enrich phosphopeptides was evaluated. In a next step, the enrichment of phosphopeptides from a whole-cell lysate of human embryonic kidney (HEK) 293T cells was performed. The eluates were analyzed using MALDI-MS in the first case and with nano-HPLC-ESI-MS/MS in the second case.The results showed that the MIPs provided affinity for phosphopeptides, binding preferentially to multi-site phosphorylated peptides. The MIPs could enrich phosphopeptides in over 10-fold compared with the number of phosphopeptides found in a cell lysate without enrichment. MS-based studies of protein phosphorylation are dependent on sample preparation steps that enrich the phosphorylated peptides prior to analysis.

Hydrophobic fumed silica dispersions in organic monomers were explored as a pore-forming system in polymer synthesis. The method developed provides a simple and effective way of controlling the pore size in highly cross-linked polymers. Fumed silica suspensions in divinylbenzene were polymerized with subsequent etching of the silica particles, therefore creating the porosity in the polymer. The resulting polymers are mesoporous materials, exhibiting an extremely narrow pore size distribution with an average pore size of about 100 Å, replicating the size of the nanofiller. BET surface areas were found appreciably high (∼350 m2/g). Furthermore, the rheological behavior of the prepolymerization mixtures was studied to elucidate the formation of the porous network and showed that a tridimensional network of particles is formed at a minimum silica fraction (Φ v ) of 0.08.

Molecularly imprinted polymers (MIPs) have been synthesized in the absence of a solvent using fumed silica nanoparticles to create a porous network. The method employed led to a chiral imprinting effect and allowed for an excellent control over the internal morphology of imprinted and non-imprinted polymer (NIP) materials. The polymers possess high surface areas (>300 m 2 ) and identical pore size (112 Å ). The MIP exhibited an imprinting factor (IF) of 9 and a selectivity value (a) of 1.83 for (2)-ephedrine.

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