Proteoform-specific enrichment of phosphopeptide isomers by polymer-based synthetic receptors

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.