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Related Experiment Video

Updated: Apr 26, 2026

Purification of Hsp104, a Protein Disaggregase
07:17

Purification of Hsp104, a Protein Disaggregase

Published on: September 30, 2011

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Probing SH2-domains using Inhibitor Affinity Purification (IAP).

Michael Höfener1, Stephanie Heinzlmeir2,3,4, Bernhard Kuster2,5

  • 1Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany.

Proteome Science
|July 29, 2014
PubMed
Summary

A novel dipeptide-derived probe effectively enriches 22 out of 50 Src homology 2 (SH2) proteins from cell lysates. This advancement aids in studying SH2 proteins and evaluating inhibitors of phosphoinositide 3-kinase (PI3K) complexes.

Keywords:
Chemical proteomicsInhibitor affinity purificationMass spectrometryPI3 kinaseSH2 domain

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Signal transduction dysregulation is linked to numerous human diseases.
  • Src homology 2 (SH2) domains, crucial for signal transduction, bind phosphotyrosine residues.
  • Investigating SH2 proteins is vital for diagnostics and medicinal chemistry, yet current methods lack comprehensive SH2 protein coverage.

Purpose of the Study:

  • To develop a single affinity method for enriching diverse SH2 proteins.
  • To create a dipeptide-derived probe for simultaneous SH2 protein investigation.
  • To assess the probe's utility in evaluating phosphoinositide 3-kinase (PI3K) complex inhibitors.

Main Methods:

  • Design and synthesis of a novel dipeptide-derived probe.
  • Evaluation of the probe's enrichment capabilities using mixed cell lysates.
  • Depletion and competition experiments to characterize SH2 binding properties.

Main Results:

  • The probe successfully enriched 22 out of 50 SH2 proteins from mixed cell lysates.
  • Demonstrated ability to enrich complexes involving SH2-bearing regulatory PI3K subunits.
  • Confirmed enrichment of catalytic phosphoinositide 3-kinase (PI3K) subunits lacking SH2 domains.

Conclusions:

  • The dipeptide-derived probe is a promising foundation for a mixed affinity resin with complete SH2 protein coverage.
  • The probe serves as a valuable tool for assessing inhibitors targeting PI3K complex disruption.
  • Enables more comprehensive investigation of SH2 protein families in future research.