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Related Experiment Video

Updated: Jul 8, 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

Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis.

Greg T Cantin¹, Wei Yi, Bingwen Lu

¹Departments of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Journal of Proteome Research

|January 29, 2008

Summary

This study enhances phosphopeptide enrichment using sequential immobilized metal affinity chromatography (IMAC) methods. Combining protein- and peptide-based IMAC with MudPIT significantly improves quantitative phosphoproteomics analysis.

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Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Published on: August 18, 2017

Area of Science:

Proteomics
Cellular signaling
Biochemistry

Background:

Immobilized metal affinity chromatography (IMAC) is widely used for phosphopeptide enrichment.
Standard IMAC struggles with complex protein mixtures, limiting its efficiency.
A need exists for improved methods to analyze phosphoproteomes in complex biological samples.

Purpose of the Study:

To evaluate the efficacy of protein-based IMAC as a pre-enrichment step before peptide-based IMAC.
To develop an optimized workflow for quantitative phosphoproteomics.
To identify phosphopeptides in the epidermal growth factor (EGF) signaling pathway.

Main Methods:

Sequential application of protein-based IMAC followed by peptide-based IMAC.
Multidimensional protein identification technology (MudPIT) for peptide separation and analysis.
Quantitative phosphoproteomic analysis of mammalian cell extracts.

Main Results:

The combined IMAC strategy significantly enhanced phosphopeptide identification.
Identified 4470 unique phosphopeptides and 4729 phosphorylation sites.
Successfully applied the method to study the epidermal growth factor pathway.

Conclusions:

Sequential IMAC enrichment is a powerful approach for complex phosphoproteomics.
This method improves the depth and accuracy of quantitative phosphoproteomic studies.
The findings provide insights into the EGF signaling pathway's phosphoregulation.