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.
Highlights
Protein phosphorylation is one of the most widely studied and best-understood post-translational modifications (PTMs)
We report on a method for enrichment of pHis peptides using custom-made molecularly imprinted polymers (MIPs)
We previously showed that imprinted polymers (MIPs) prepared using urea-based N-3,5-bis(trifluoromethyl)-phenyl-N′-4-vinylphenylurea, functional monomer 1 (Figure 1), display a high affinity for phosphorylated peptides[28,29] and that the selectivity for either phosphoserine or phosphotyrosine peptides can be programmed using the appropriate template.[30−32] Mechanistically, the hydrogen bond-driven recognition and the charge-neutral resin character distinguish these phases from currently used phosphoenrichment tools, e.g., IMAC, TiO2, and antibodies, and could explain the reduced chargedependent sequence bias of the enriched phosphopeptide pool
Summary
Protein phosphorylation is one of the most widely studied and best-understood post-translational modifications (PTMs) It is involved in the regulation of vital biological processes, including cellular signal transduction.[1] phosphorylation can occur on at least nine different amino acid residues, i.e., serine, threonine, tyrosine, histidine, lysine, arginine, aspartate, glutamate, and cysteine, the vast majority of phosphoproteomics research is focused on the phosphorylation of the former three residues. The role of histidine phosphorylation (pHis) has gained attention This modification is well known to be involved in two-component protein-signaling networks in prokaryotes and lower eukaryotes[2−4] and is found in mammals and implicated in certain human disease states.[5−7]. It has been estimated that histidine phosphorylation in eukaryotes accounts for 6% of the total protein phosphorylation[9] and is more abundant than the phosphotyrosine (pTyr) modification
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