Analytical Chemistry

Sequence Specific Phosphopeptide Enrichment of ZAP-70 regulatory motifs using epitope-imprinted polymer complements

Authors

Abstract

Immunoaffinity enrichment based on antipeptide antibodies coupled to mass spectrometry-based identification and quantification (immuno-MS) is a promising approach to translate proteomics to clinical assays with diagnostic value. This is linked to precision cancer medicine, where immuno-MS based studies of protein phosphorylation dependent-signalling states of cells enable pathway targeted therapies and response monitoring to be developed. Clinically robust methods depend on the availability of high quality phosphopeptide antibodies but these rarely meet the stringent demands on reproducibility, robustness, availability and peptide specificity. We here show that polymer-based “plastic antibodies” prepared by molecular imprinting could play this role. Focusing on the Tyr-492 and Tyr-493 kinase regulatory motif of the SH2 domain in ZAP-70, a critical mediator in T-cell receptor signalling, we show that MIPs can be easily generated to recognize corresponding mono- or di- phosphorylated tryptic peptides in a highly specific manner. The polymers bound their complementary phosphorylated decapeptides with Kd:s in the low µM range in both low aqueous proteomics media and aqueous buffers. Moreover, only minor cross-reactivity was observed for phosphopeptide variants with identical amino acid sequence. Proving their practical value, we demonstrate their use for sequence specific phosphopeptide enrichment from extracts of Jurkat T-cells stimulated to induce protein phosphorylation at either of the two regulatory sites of ZAP-70. We expect the approach to be broadly applicable paving the way for robust, low-cost multiplex phosphopeptide assays.

Content

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Supplementary material

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Supporting Information
Reagents, apparatus and methods, synthesis and characterisation of MIP binders, cell culture, lysis and enrichment experiments.