Chemical tools for studying phosphohistidine: generation of selective τ-phosphohistidine and π-phosphohistidine antibodies
Abstract Nonhydrolysable stable analogues of τ-phosphohistidine (τ-pHis) and π-pHis have been designed, aided by electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as hapte...
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| Main Authors: | , , , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
August 15, 2023
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| In: |
ChemBioChem
Year: 2023, Volume: 24, Issue: 16, Pages: 1-15 |
| ISSN: | 1439-7633 |
| DOI: | 10.1002/cbic.202300182 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1002/cbic.202300182 Verlag, lizenzpflichtig, Volltext: https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202300182 
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| Author Notes: | Mehul V. Makwana, Cleide dos Santos Souza, Barry T. Pickup, Mark J. Thompson, Santosh K. Lomada, Yuxi Feng, Thomas Wieland, Richard F. W. Jackson, and Richmond Muimo |
| Summary: | Abstract Nonhydrolysable stable analogues of τ-phosphohistidine (τ-pHis) and π-pHis have been designed, aided by electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by 31P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA?G-τ-pHis and BSA?G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the pHis antibodies generated by using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated by using phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated by using pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis. |
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| Item Description: | Online veröffentlicht: 14. Mai 2023 Gesehen am 16.10.2024 |
| Physical Description: | Online Resource |
| ISSN: | 1439-7633 |
| DOI: | 10.1002/cbic.202300182 |