Related Experiment Video
Updated: Jul 7, 2026
09:16
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Phosphopeptide mapping and identification of phosphorylation sites
J Meisenhelder1, T Hunter, P van der Geer
1The Salk Institute for Biological Studies, La Jolla, California, USA.
Current Protocols in Molecular Biology
|February 12, 2008
Summary
This study details a method for analyzing protein phosphorylation sites using phosphopeptide mapping. This technique visualizes phosphorylated peptides to identify and quantify phosphorylation sites on proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Protein phosphorylation is a critical post-translational modification regulating cellular processes.
- Understanding phosphorylation site dynamics is essential for deciphering signaling pathways.
Purpose of the Study:
- To describe a detailed procedure for proteolytic digestion of radiolabeled proteins.
- To establish phosphopeptide mapping for analyzing phosphorylation sites.
- To enable identification of phosphorylation sites and their changes upon cellular treatments.
Main Methods:
- Proteolytic digestion of 32P-labeled proteins.
- Two-dimensional thin-layer chromatography (TLC) for phosphopeptide separation.
- Autoradiography for phosphopeptide visualization.
- High-performance liquid chromatography (HPLC) for purification.
- Mass spectrometry and peptide microsequencing for analysis.
Main Results:
- Phosphopeptide maps provide information on the number of phosphorylation sites.
- The method allows detection of changes in phosphorylation sites.
- Identification of specific phosphorylation sites is achievable.
Conclusions:
- Phosphopeptide mapping is a valuable tool for studying protein phosphorylation.
- This technique aids in understanding protein function and regulation.
- Combined with advanced analytical methods, it offers comprehensive insights into phosphorylation dynamics.
Related Concept Videos
02:54Protein Kinases and Phosphatases
Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:02Phosphorylation
The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
01:02Phosphorylation
The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
01:33Peptide Identification Using Tandem Mass Spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...