Phosphate Group-directed Molecular Imprinting And Its Application For Protein Phosphorylation

Phosphorylation,as one of the most important post translational modifications,plays a vital role in signal transduction,cell division,cytoskeleton dynamics and metabolism.Protein phosphorylation mainly occurs on the amino acids of Ser,Thr and Tyr with a relative ratio of 1800:200:1.Abnormal phosphorylation is associated with illness and is often used as a biomarker.Mass spectrometry-based proteomics provides a very important tool for phosphorylated protein analysis.Apart from the difficulty of recognizing peptidome in physiological samples,the poor ionization efficiency and low concentration of phosphopeptides,as well as the significant interferences caused by nonphosphopeptides,make direct mass spectrometry-based phosphopeptidome more difficult.Although there are ready enrichment approaches towards different phosphorylation forms,there is lacking a method targeting only one selected phosphopeptide.Molecularly imprinted polymers(MIPs)are engineered recognizing tools that are capable of binding to a specific molecule through functional modification.Nowadays,MIPs are widely used in various fields,such as sensing,separation,catalysis,disease diagnosis,etc.;due to their easy preparation,good stability and low cost,MIPs have recently been involuting as a replacement for antibodies in the research of phosphoproteomics.In this paper,nanomaterials prepared by molecular imprinting technology are used for selective identification and enrichment of phosphorylated peptides,which mainly include the following three aspects:1.The paper firstly verified the reliability of the imprinting material preparation method by using the principle of directional imprinting,three molecular imprinting materials were prepared using cytidine monophosphate as the template,and the static adsorption curves of these three synthesized MIPs and NIPs on small molecule cytidine monophosphate were compared respectively,and the results showed that the best imprinting effects were obtained for the directional imprinting as well as the template washing prepared materials,which were also applicable to the subsequent peptide and protein enrichment systems.2.By combining Ti O₂and molecular imprinting with single phosphopeptide as template,UPTES as functional monomer and TEOS as cross-linker,the two affinities can work together to enhance the binding affinity to specific phosphopeptides for specific recognition of peptide sequences,and MIPs synthesized in this way are applied for precise recognition of peptides with different phosphorylated forms(serine,threonine,tyrosine).The precise identification of individual phosphopeptides with different phosphorylated forms(serine,threonine,tyrosine)was achieved by MALDI-TOF MS detection of MIPs enriched for standard peptide mixtures and tryptic digests of casein/milk.3.Using the principle of directional epitope imprinting,phosphorylated peptides with similar sequences to proteins are used as"epitopes"to specifically identify target proteins.In this paper,we used complete phosphopeptides as templates,and formed peptide cavities containing homologous sequences with target proteins in MIPs after material elution,and used this"epitope"to specifically identify?-casein and HSA.