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Updated: Jul 3, 2026

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Published on: November 7, 2019

A parallel affinity purification method for selective isolation of polyubiquitinated proteins.

Kazuhisa Ota¹, Keiji Kito, Shun-ichiro Iemura

¹Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan.

|July 11, 2008

Summary

We developed a parallel affinity purification (PAP) method to selectively enrich ubiquitinated proteins. This technique improves the identification of ubiquitinated proteins using mass spectrometry.

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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Published on: March 23, 2020

Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Ubiquitination is a crucial post-translational modification regulating numerous cellular processes.
Identifying ubiquitinated proteins is essential for understanding their functions.
Existing methods often struggle with abundant non-ubiquitinated proteins and low-abundance ubiquitinated targets.

Purpose of the Study:

To develop a novel purification method for selectively enriching ubiquitinated proteins.
To enhance the efficiency and accuracy of identifying ubiquitinated proteins via mass spectrometry.

Main Methods:

Developed a parallel affinity purification (PAP) procedure.
Coexpressed two affinity-tagged ubiquitins in cells.
Utilized sequential affinity chromatography specific to each tag.

Main Results:

PAP effectively eliminates ubiquitin monomers and monoubiquitinated proteins.
Achieved selective enrichment of poly- and multiubiquitinated proteins.
Demonstrated improved identification of ubiquitinated proteins.

Conclusions:

The PAP procedure is a powerful tool for studying ubiquitination.
This method facilitates the mass-spectrometric identification of ubiquitinated proteins.
PAP offers a significant advancement over previous purification techniques.