Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment

Area of Science:

• Biochemistry
• Molecular Biology
• Gerontology

Background:

• The transmembrane protein α-Klotho is expressed in the kidney, parathyroid gland, and choroid plexus.
• Reduced α-Klotho levels are linked to chronic kidney disease and other age-related conditions.
• Detecting soluble α-Klotho in biofluids has been a significant challenge in research.

Purpose of the Study:

• To discover high-affinity and selective peptide binders for the α-Klotho protein.
• To develop novel tools for detecting and studying soluble α-Klotho.
• To advance research into the physiological roles of α-Klotho, including its potential hormonal function.

Main Methods:

• Employed in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS) for peptide discovery.
• Utilized two rounds of AS-MS followed by N-terminal modifications to optimize peptide binders.
• Compared the binding affinity of newly discovered peptides against a previously designed peptide (Pep-10).

Main Results:

• Discovered novel peptides with significantly improved binding affinity (approx. 2300-fold increase) to α-Klotho compared to Pep-10.
• Demonstrated the ability of lead peptide binders to enrich α-Klotho from cell lysates.
• Showcased successful labeling of α-Klotho within kidney cells using the developed peptide probes.

Conclusions:

• The study successfully identified high-affinity, selective peptide binders for α-Klotho using AS-MS.
• These peptide binders serve as functional probes, enabling better detection and study of α-Klotho in biological samples.
• Validated the utility of label-free, in-solution AS-MS for discovering peptide binders for challenging protein targets.