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

Peiyuan Zhang1, Xiyun Ye1, John C K Wang2

  • 1Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

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Summary

Researchers discovered new peptides that bind strongly to the aging-related protein α-Klotho. This breakthrough aids in detecting soluble α-Klotho, crucial for understanding age-related diseases and Klotho

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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.