Site-level resolution of protein phosphorylation remains a central challenge in decoding cellular signaling and conventional global phosphoproteomics is limited in its ability to detect low-abundance peptides and resolve positional isomers. Here we introduce sequence-selective synthetic receptors based on imprinted polymers for targeted enrichment of defined phosphopeptide motifs from complex cancer cell proteomes. By encoding local sequence context into the binding interface, these receptors enable selective capture of closely related phosphorylation sites with high specificity. Using the T cell kinase ZAP70 as a model, we resolve phosphorylation at adjacent tyrosine residues (pY492 and pY493) within a regulatory motif, overcoming a long-standing limitation in site discrimination. Integrated with liquid chromatography–mass spectrometry workflows, this enables sensitive detection of low-abundance and isomeric phosphopeptides that evade conventional enrichment strategies. More broadly, our findings establish molecular imprinting as a programmable chemical platform for site-resolved phosphoproteomics, opening new avenues to interrogate signaling networks with molecular precision.

Protein phosphorylation is a reversible post-translational modification (PTM) acting as a key regulator of protein function with an impact on cell behavior and pathological conditions. Accurate monitoring of protein phosphorylation status is important for both mechanistic deciphering of cellular processes and disease diagnostics. However, detecting this PTM remains challenging due to its transient nature, stability issues, and low abundance. Molecularly imprinted polymers (MIPs) fill a void in the search for robust affinity techniques that can be tailored to specifically enrich phospho-peptides at predetermined levels of discrimination. This ranges from (i) surface-imprinted mesoporous materials for generic enrichment; (ii) amino acid–specific receptors that fractionate by side chain with minimal sequence bias; (iii) dummy-templated MIPs for labile phosphorylations; to (iv) sequence-specific receptors for defined phospho-epitopes and biomarkers. This review offers a concise, interdisciplinary overview of the field in the context of current challenges in phosphoproteomics.

Early detection of cancer is essential for successful treatment and improvement in patient prognosis. Deregulation of post-translational modifications (PTMs) of proteins, especially phosphorylation, is present in many types of cancer. Therefore, the development of materials for the rapid sensing of low abundant phosphorylated peptides in biological samples can be of great therapeutic value. In this work, we have synthesised fluorescent molecularly imprinted polymers (fMIPs) for the detection of the phosphorylated tyrosine epitope of ZAP70, a cancer biomarker. The polymers were grafted as nanometer-thin shells from functionalised submicron-sized silica particles using a reversible addition-fragmentation chain-transfer (RAFT) polymerisation. Employing the combination of fluorescent urea and intrinsically cationic bis-imidazolium receptor cross-linkers, we have developed fluorescent sensory particles, showing an imprinting factor (IF) of 5.0. The imprinted polymer can successfully distinguish between phosphorylated and non-phosphorylated tripeptides, reaching lower micromolar sensitivity in organic solvents and specifically capture unprotected peptide complements in a neutral buffer. Additionally, we have shown the importance of assessing the influence of counterions present in the MIP system on the imprinting process and final material performance. The potential drawbacks of using epitopes with protective groups, which can co-imprint with targeted functionality, are also discussed.

The use of polymerizable hosts in anion imprinting has led to powerful receptors with high oxyanion affinity and specificity in both aqueous and non-aqueous environments. As demonstrated in previous reports, a carefully tuned combination of orthogonally interacting binding groups, for example, positively charged and neutral hydrogen bonding monomers, allows receptors to be constructed for use in either organic or aqueous environments, in spite of the polymer being prepared in non-competitive solvent systems. We here report on a detailed experimental design of phenylphosphonic and benzoic acid-imprinted polymer libraries prepared using either urea-or thiourea-based host monomers in the presence or absence of cationic comonomers for charge-assisted anion recognition. A comparison of hydrophobic and hydrophilic crosslinking monomers allowed optimum conditions to be identified for oxyanion binding in non-aqueous, fully aqueous, or high-salt media. This showed that recognition improved with the water content for thiourea-based molecularly imprinted polymers (MIPs) based on hydrophobic EGDMA with an opposite behavior shown by the polymers prepared using the more hydrophilic crosslinker PETA. While the affinity of thiourea-based MIPs increased with the water content, the opposite was observed for the oxourea counterparts. Binding to the latter could however be enhanced by raising the pH or by the introduction of cationic amine-or Na+-complexing crown ether-based comonomers. Use of high-salt media as expected suppressed the amine-based charge assistance, whereas it enhanced the effect of the crown ether function. Use of the optimized receptors for removing the ubiquitous pesticide glyphosate from urine finally demonstrated their practical utility.

Antibodies have become a critical component of many diagnostic assays and are used for therapeutic purposes. Nevertheless they often fail to meet the strict performance demands raised in industry and in the clinic (e.g. stability, reproducibility, selectivity, affinity). These issues are especially notable for assays targeting post translational modifications (PTM) of proteins (phosphorylation, glycosylation, sulfation etc.). Antibody-based technologies suffer from problems of a more general nature associated with the analytical use of biological receptors i.e.: i) limited stability requiring cold chain logistics, ii) high production costs, iii) batch to batch variability. The above emphasizes the need for alternative robust, reproducible and low cost “binders” and assays. The aim in this thesis is to design, develop and test molecularly imprinted polymers (MIPs) which were synthesized epitope and stoichiometric imprinting approaches targeting phosphorylation as a PTM. Protein phosphorylationis one of the most common PTM, which is based on covalent attachment of phosphate group to particular amino acids. Misregulation of phosphorylation process is found related with diseases such as cancer, diabetes, and neurodegeneration. MIPs are synthesized through copolymerization of functional monomers and crosslinkers in the presence of N- and C- terminal protected templates. The key recognition element employed in developed synthetic receptors was 1,3-diaryl urea functionalmonomer 1. This monomer is a potent hydrogen bond donor forming strong cyclichydrogen bonds with oxyanions. Amino acid sequence specific and side chain imprinted binders were prepared targeting phosphorylation on tyrosine (pTyr) and on histidine (pHis). pHis MIP-based approach is proposed as a solution to enrich pHis peptides in the presence of other phosphoesters such as phosphoserine (pSer) in complex mixture without pre-treatment like β-elimination. In pTyr, ZAP-70 (zeta associated 70 kDa protein), which is prognosticator for chronic lymphocytic leukemia (CLL), and pTyr-sequence specific motif Src-SH2 domain were chosen as targets to evaluate regio- or stoichiometric selectivity performance of imprinted polymers. The synthesized polymers are used as effective enrichment tools for target phosphorylated peptides from complex mixture prior to mass spectrometry. Overall, the results demonstrate unique proteomics enrichment tools that link with personalized medicine relying on diagnostic coupled cancer treatment strategies based on kinase inhibitors.

The field of anion recognition in aqueous media is dominated by multiple positive charges and/or metal ions containing synthetic receptors. Here, we report the synthesis of a highly water-compatible oxyanion-imprinted neutral receptor by cross-linking the polymerizable diary! urea host monomer and phenyl phosphonic acid (PPA) complex in the presence of a large number of comonomers containing hydroxyl or methyl ester groups. We have demonstrated that the affinity of the imprinted receptor for anions is easily tuned by decorating the hydrophilic and hydrophobic end groups around the binding pocket of the imprinted receptor. The binding characteristics of the synthesized artificial binders were evaluated in aqueous and nonaqueous environments. We have demonstrated practical applicability of the PPA-imprinted receptor by packing in the solid-phase extraction cartridge which was used to separate the ubiquitous pesticide glyphosate from water. Also, the developed receptor can selectively bind to phosphorylated tyrosine amino acids and phosphorylated tyrosine octapeptides in aqueous buffered media than to other phosphorylated and nonphosphorylated analogues. The approach utilized here to use an inexpensive dummy template can be utilized for the synthesis of imprinted receptors for anions with phosphate motifs.

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and β-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.

The design of hosts for either cations or anions is complicated due to the competition for binding by the host or guest counterions. Imprinting relying on self-assembly offers the possibility to stabilize the guest and its counterion in a favorable geometry. We here report on a comprehensive supramolecular approach to anion receptor design relying on concurrent recognition of both anion and cation. This was achieved by high order complex imprinting of the disodium salt of phenyl-phosphonic acid in combination with neutral urea and sodium ion selective 18-crown-6 monomers. The polymers displayed enhanced affinity for the template or inorganic phosphate or sulfate in competitive aqueous buffers, with affinity and selectivity increasing with increasing ionic strength. The presence of engineered sites for both ionic species dramatically increases the salt uptake in strongly competitive media such as brine.