Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers

Abstract

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.

Enzymology and significance of protein histidine methylation

“…Moreover, His phosphorylation is a labile PTM that is lost during standard phosphoproteomics workflows and is thus scientifically underexplored ( 80 ). New tailored workflows for His phosphorylation proteomics enable both stabilization and affinity enrichment of the PTM ( 81 ). Future studies will likely explore the overlap of His phosphorylation and methylation sites and elucidate whether the PTMs interplay through competition.…”

“…Another approach to distinguish 1MeH and 3MeH would involve enrichment using isomer-specific affinity agents before MS analysis. Such affinity agents are not yet available, but recent reports have demonstrated the generation of molecularly imprinted polymers to enrich His phosphorylated peptides for proteomics ( 81 ), an approach also applicable to Hme. The generation of polyclonal antibodies through rabbit immunization has also been proven feasible for His phosphorylation ( 94 , 95 ), but also not yet applied to Hme.…”

Aggregation-induced emission monomer-based fluorescent molecularly imprinted poly(ionic liquid) synthesized by a one-pot method for sensitively detecting 4-nitrophenol

“…Molecularly imprinted polymers (MIPs) are customized materials that exhibit recognition sites for template molecules. 9–12 Fluorescent MIPs can be primarily synthesized by modifying MIPs with fluorescent substances to detect different analytes. 13–15 Among numerous fluorescent MIP synthesis techniques, the one-pot polymerization strategy using polymerized fluorescent monomers to prepare fluorescent MIPs is considered as a simple preparation process.…”

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

“…MIPs targeting protein PTMs have in this context emerged as a promising alternative to established affinity reagents. 24-29 30 31-33 For instance, efficient amino acid side chain specific enrichment of tryptic peptides from cell lysates or complex protein digests have been demonstrated for phosphotyrosine 31 , phosphoserine 27 and most recently labile phosphohistidine PTMs 33 . The design of these MIPs relies on the use of neutral urea-based host monomers with tunable affinity for oxyanions such as carboxylates and phosphates.…”