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Published on: February 28, 2015
Peptide aptamers in label-free protein detection: 2. Chemical optimization and detection of distinct protein isoforms
Jason J Davis1, Jan Tkac, Rachel Humphreys
1Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA.
Abstract:
The early detection and diagnosis of cancer lies central to successful treatment and improved patient outcome. Current techniques are limited by the nature of the biological receptor and the assays available. This paper reports the use of novel biological probes, peptide aptamers, in detecting cyclin-dependent protein kinases (CDKs) whose activity is important in proliferating and cancerous cells. We describe, specifically, the optimization of an orientated peptide aptamer surface and its utilization in establishing a highly specific, low-nanomolar sensitive, detection protocol for the active form of CDK2. In comparing target binding affinity of two different aptamers (pep6 and pep9), both constructed through the insertion of peptide sequences into the surface of a scaffold protein, one was observed to be consistently more effective. Significantly, the pep9 aptamers were able to detect subtle changes in the conformation of CDK2 associated with activation of its catalytic activity that may be caused by the phosphorylation of a single amino acid (threonine 160). A typical response toward the inactive form of CDK2 was in the range of 0.5-2% of the binding of the active form of CDK2 in the concentration range from 2 to 20 nM. Although antibodies are occasionally able to recognize conformations in their targets, this is the first time that a nonantibody protein probe has been used to detect an active protein isoform. Because peptide aptamers are usually raised against full-length proteins, this raises the possibility that peptide aptamers will be able to extend the repertoire of probes that recognize protein conformations, post-translational modifications (PTMs), or conformations stabilized by PTMs.
Insights
Novel peptide aptamers offer a new method for early cancer detection by identifying active cyclin-dependent protein kinases (CDKs). This breakthrough enables sensitive detection of active CDK2, improving cancer diagnosis and patient outcomes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Early cancer detection is crucial for successful treatment and patient outcomes.
- Current diagnostic techniques face limitations due to biological receptor and assay constraints.
- Cyclin-dependent protein kinases (CDKs) are vital in cell proliferation and cancer development.
Purpose of the Study:
- To develop novel biological probes for detecting active cyclin-dependent protein kinases (CDKs).
- To optimize a peptide aptamer surface for highly specific and sensitive detection of active CDK2.
- To explore the potential of peptide aptamers in identifying specific protein conformations and post-translational modifications.
Main Methods:
- Utilized peptide aptamers as novel biological probes.
- Optimized an orientated peptide aptamer surface for detection assays.
- Compared the binding affinity of two distinct peptide aptamers (pep6 and pep9) against active CDK2.
- Assessed the ability of aptamers to detect conformational changes in CDK2.
Main Results:
- Achieved low-nanomolar sensitivity in detecting the active form of CDK2.
- Identified pep9 aptamers as more effective in binding target CDK2.
- Demonstrated that pep9 aptamers can detect subtle conformational changes in CDK2 associated with catalytic activation, including phosphorylation at threonine 160.
- Observed minimal binding (0.5-2%) of inactive CDK2 compared to active CDK2 in the 2-20 nM range.
Conclusions:
- Peptide aptamers represent a novel, non-antibody protein probe for detecting active protein isoforms.
- This study demonstrates the first use of a non-antibody protein probe to identify an active protein isoform.
- Peptide aptamers show promise in expanding the range of probes for recognizing protein conformations, PTMs, or PTM-stabilized conformations, aiding in cancer diagnostics.
