Protein phosphorylation is a reversible posttranslational modification (PTM) playing a central role in numerous biological events including disease pathogenesis. Thus analysis of phosphoproteome is crucial for understanding cellular regulation processes and can facilitate the development of new diagnostic and therapeutic tools. Difficulties in analysis of phosphoproteome arise from low abundance and transient apparence of phosphoproteins. This creates a need for effective enrichment tools for phospho- proteins and peptides prior to mass spectrometry (MS) analysis.<br/>We have previously reported on the succesfull development of phosphotyrosine selective MIPs (pY-MIP) as crushed monoliths. In order to integrate these receptors in high throughput enrichment platforms, polymers in beaded format are prefered (Figure 1). Apart from controlling the particle size and shape an effective pore size control is required in order to avoid size dependent bias in the peptide capture step. Here we have investigated templated synthesis and surface imprinting to address these issues. We show that by using the simple approach of polymerization inside pores of porous silica the pY-MIP could be synthesized in microspherical format while preserving selectivity and binding properties of the crushed monoliths. Apart from improved yield and simplification of polymer processing this approach allows the control of polymer size, shape and porosity. Furthermore, we prepared pY-MIP by hierarchical imprinting where the template is immobilized on the silica surface prior to polymerization. The obtained materials show enhanced selectivity for phosphotyrosine containing peptides and a capacity scaling with template load. Tests of the beads for the enrichment of phosphopeptides from tryptic digests of twelve proteins revealed both pY/pS and pY/Y selectivity.

Surface imprinted phosphopeptide capture beads

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.