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Related Experiment Video

Updated: Jun 13, 2026

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Published on: August 2, 2018

Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples.

Juanying Ye¹, Xumin Zhang, Clifford Young

¹Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

Journal of Proteome Research

|May 11, 2010

Summary

This study optimizes immobilized metal ion affinity chromatography (IMAC) for phosphopeptide enrichment, enhancing efficiency and specificity in large-scale phosphoproteomics. The improved Fe(III)-NTA IMAC method enables deeper characterization of phosphoproteins.

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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

Area of Science:

Proteomics
Biochemistry
Analytical Chemistry

Background:

Immobilized metal ion affinity chromatography (IMAC) is a common method for phosphopeptide enrichment.
However, its robustness, efficiency, and specificity in large-scale phosphoproteomics are debated.

Purpose of the Study:

To compare IMAC materials and optimize protocols for enhanced phosphopeptide enrichment.
To investigate factors influencing IMAC performance and improve phosphoproteome analysis.

Main Methods:

Compared three IMAC materials under varying conditions, selecting Fe(III)-nitrilotriacetic acid (NTA) resin.
Investigated ionization efficiency changes with acetonitrile concentration.
Developed an optimized Fe(III)-NTA IMAC protocol and applied it to complex biological samples.

Main Results:

Fe(III)-NTA IMAC showed superior performance; higher iron purity increased enrichment efficiency.
The optimized protocol achieved highly selective phosphopeptide enrichment, even from diluted samples.
Over 1000 phosphorylation sites were identified from mouse and Drosophila melanogaster samples using the improved IMAC-IMAC method and LC-MS/MS.
Demonstrated efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides.

Conclusions:

Rational improvements to the IMAC protocol enhance phosphopeptide recovery and specificity.
The optimized IMAC-IMAC method allows for more detailed phosphoprotein characterization in functional phosphoproteomics.