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Updated: Dec 9, 2025
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Functional Nanochannels for Sensing Tyrosine Phosphorylation
Minmin Li1,2, Yuting Xiong1,2, Wenqi Lu1,3
1CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P. R. China.
Abstract:
Tyrosine phosphorylation (pTyr), much of which occurred on localized multiple sites, initiates cellular signaling, governs cellular functions, and its dysregulation is implicated in many diseases, especially cancers. pTyr-specific sensing is of great significance for understanding disease states and developing targeted anticancer drugs, however, it is very challenging due to the slight difference from serine (pSer) or threonine phosphorylation (pThr). Here we present polyethylenimine-g-phenylguanidine (PEI-PG)-modified nanochannels that can address the challenge. Rich guanidinium groups enabled PEI-PG to form multiple interactions with phosphorylated residues, especially pTyr residue, which triggered the conformational change of PEI-PG. By taking advantage of the "OFF-ON" change of the ion flux arising from the conformational shrinkage of the grafted PEI-PG, the nanochannels could distinguish phosphorylated peptide (PP) from nonmodified peptide, recognize PPs with pSer, pThr, or pTyr residue and PPs with different numbers of identical residues, and importantly could sense pTyr peptides in a biosample. Benefiting from the strong interaction between the guanidinium group and the pTyr side-chain, the specific sensing of pTyr peptide was achieved by performing a simple logic operation based on PEI-PG-modified nanochannels when Ca2+ was introduced as an interferent. The excellent pTyr sensing capacity makes the nanochannels available for real-time monitoring of the pTyr process by c-Abl kinase on a peptide substrate, even under complicated conditions, and the proof-of-concept study of monitoring the kinase activity demonstrates its potential in kinase inhibitor screening.
Insights
New nanochannels modified with polyethylenimine-g-phenylguanidine (PEI-PG) can specifically detect tyrosine phosphorylation (pTyr) in biological samples. This breakthrough enables precise sensing of pTyr peptides, crucial for cancer research and drug development.
Area of Science:
- Biotechnology and Nanotechnology
- Molecular Biology and Biochemistry
- Cancer Research
Background:
- Tyrosine phosphorylation (pTyr) is a critical regulator of cellular signaling, governing cell functions, and its dysregulation is linked to diseases, particularly cancers.
- Accurate sensing of pTyr is vital for understanding disease mechanisms and developing targeted anticancer therapies, but it is challenging due to its similarity to serine (pSer) and threonine phosphorylation (pThr).
- Existing methods face difficulties in distinguishing pTyr from other phosphorylation types, hindering precise molecular diagnostics and therapeutic development.
Purpose of the Study:
- To develop a novel sensing platform capable of specifically detecting tyrosine phosphorylation (pTyr) in complex biological environments.
- To overcome the challenge of differentiating pTyr from pSer and pThr using a nanotechnology-based approach.
- To demonstrate the utility of the developed sensor for monitoring kinase activity and screening kinase inhibitors.
Main Methods:
- Modification of nanochannels with polyethylenimine-g-phenylguanidine (PEI-PG) to enhance interactions with phosphorylated residues.
- Utilizing the conformational change of PEI-PG upon binding to phosphorylated peptides, leading to an 'OFF-ON' ion flux signal.
- Implementing a logic operation with Ca2+ as an interferent to achieve specific pTyr sensing.
Main Results:
- The PEI-PG-modified nanochannels successfully distinguished phosphorylated peptides from non-modified peptides.
- The nanochannels demonstrated the ability to recognize peptides with pSer, pThr, or pTyr residues, and peptides with varying numbers of identical residues.
- Specific sensing of pTyr peptides was achieved with high accuracy, even in the presence of Ca2+ as an interferent, and real-time monitoring of c-Abl kinase activity on a peptide substrate was demonstrated.
Conclusions:
- Polyethylenimine-g-phenylguanidine (PEI-PG)-modified nanochannels offer a robust and specific platform for sensing tyrosine phosphorylation (pTyr).
- The developed sensing technology enables precise detection of pTyr peptides in biosamples and real-time monitoring of kinase activity.
- This approach holds significant potential for advancing cancer diagnostics, understanding disease states, and facilitating the screening of kinase inhibitors.
