Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Author Correction: Listeria monocytogenes impairs SUMOylation for efficient infection.

Nature·2020

Same author

A new evolutionary model for the vertebrate actin family including two novel groups.

Molecular phylogenetics and evolution·2019

Same author

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Nature chemical biology·2019

Same author

The Plant PTM Viewer, a central resource for exploring plant protein modifications.

The Plant journal : for cell and molecular biology·2019

Same author

EXPANSIN A1-mediated radial swelling of pericycle cells positions anticlinal cell divisions during lateral root initiation.

Proceedings of the National Academy of Sciences of the United States of America·2019

Same author

Damage on plants activates Ca<sup>2+</sup>-dependent metacaspases for release of immunomodulatory peptides.

Science (New York, N.Y.)·2019

Search research articles

Related Experiment Video

Updated: Jun 25, 2026

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Published on: October 27, 2023

Reverse-phase diagonal chromatography for phosphoproteome research.

Kris Gevaert¹, Joël Vandekerckhove

¹Department of Medical Protein Research, Ghent University, Ghent, Belgium.

Methods in Molecular Biology (Clifton, N.J.)

|February 26, 2009

Summary

This summary is machine-generated.

This study introduces a novel gel-free method using diagonal chromatography to isolate phosphorylated peptides from complex biological samples. This technique enhances phosphoproteomics research by specifically sorting modified peptides.

More Related Videos

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Published on: June 25, 2020

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Published on: May 4, 2022

Related Experiment Videos

Last Updated: Jun 25, 2026

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Published on: October 27, 2023

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Published on: June 25, 2020

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Published on: May 4, 2022

Area of Science:

Proteomics
Biochemistry
Analytical Chemistry

Background:

Phosphorylation is a critical post-translational modification regulating cellular processes.
Isolating phosphopeptides from complex proteomes is challenging due to low abundance and high complexity.
Existing methods often involve multiple steps or specialized equipment.

Purpose of the Study:

To develop a gel-free proteomics procedure for specific isolation of phosphorylated peptides.
To present the complete COFRADIC (COlumn FRActionatIon DIfferential CHemistry) sorting procedure.
To enhance phosphopeptide enrichment and identification in complex biological samples.

Main Methods:

Utilizing diagonal, reverse-phase chromatography with an intermediate dephosphorylation step.
Employing a cocktail of broad-spectrum phosphatases for targeted phosphopeptide modification.
Incorporating a pre-enrichment step and differential isotope labeling to improve yield and accuracy.

Main Results:

The procedure effectively isolates phosphorylated peptides from whole proteome digests.
Dephosphorylation causes a hydrophobic shift, enabling separation from non-phosphorylated peptides.
Differential isotope labeling distinguishes true phosphopeptides from artificially sorted ones.

Conclusions:

The described COFRADIC method provides a robust, gel-free approach for phosphopeptide isolation.
This technique advances phosphoproteomics by improving the specificity and yield of phosphopeptide enrichment.
The method facilitates deeper insights into cellular signaling pathways regulated by phosphorylation.