In-Situ Digestion Assisted Multi-Template Imprinted Nanoparticles for Efficient Analysis of Protein Phosphorylation

Abstract

Protein phosphorylation is one of the most important post-translational modifications in mammals. Since abnormal phosphorylation is often associated with the occurring of diseases, the understanding of protein phosphorylation is of great importance. Among the exiting examples of receptors for phosphorylation recognition, molecularly imprinted polymers (MIPs) have proven to be the effective alternative of the widely used biological or chemical affinity ligands. However, all the reported phospho-specific MIPs were limited to the simple templates (e.g. phosphate or phospho-amino acid). Because of the limited chemical and structural information of these simple templates, they are not favorable for preparation of high-performance MIPs. Herein, we propose a new general approach called in-situ digestion assisted multi-template imprinting for preparation of phospho-specific molecularly imprinted nanoparticles. Through the novel templating strategy and controllable imprinting process, imprinted nanoparticles specific to the intact phosphoprotein and its phosphopeptides were synthesized. In the meantime, we are also able to realize the fine tuning of the recognition at peptide level of the imprinted nanoparticles. The prepared nanoparticles exhibited excellent specificity, high affinity and good generality. Based on the selective enrichment of the imprinted nanoparticles, the MS identification of both the intact phosphoprotein and phosphopeptides in real complex samples could be achieved. Therefore, we believe that the in-situ digestion assisted multi-template imprinting strategy holds promising future in both phosphorylation analysis and proteomics applications.

Keywords: molecule imprinting, protein phosphorylation, Phosphopeptides, nanoparticles, Affinity separation