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Related Concept Videos

Phosphorylation01:02

Phosphorylation

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...

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Updated: Jun 4, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

One-source peptide/phosphopeptide standards for accurate phosphorylation degree determination.

Bettina Hahn1, Martin Böhm, Valentina Raia

  • 1Molecular Structure Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Proteomics
|January 27, 2011
PubMed
Summary

This study introduces a novel method using stable isotope-labeled standards for precise protein phosphorylation degree determination. The technique accurately quantifies phosphorylation in signaling proteins like STAT5A/B and STAT6.

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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Published on: August 17, 2015

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Last Updated: Jun 4, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
09:04

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Published on: August 17, 2015

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Reversible protein phosphorylation is crucial for intracellular signal transduction pathways.
  • Accurate quantification of phosphorylation site occupancy is essential for understanding cellular signaling.
  • Existing methods may lack the precision for site-specific phosphorylation degree determination.

Purpose of the Study:

  • To develop an innovative and accurate method for site-specific protein phosphorylation degree determination.
  • To establish a reliable internal standard for quantifying both peptide and phosphopeptide analytes.
  • To demonstrate the method's applicability on key signaling proteins.

Main Methods:

  • Utilizing nanoLC-ESI-MS/MS for high-resolution analysis.
  • Employing a stable isotope-labeled peptide/phosphopeptide standard pair with a defined molar ratio.
  • Developing a 'one-source' standard by quantitative dephosphorylation of a labeled phosphopeptide standard.
  • Mixing peptide and phosphopeptide standards at precise volumetric ratios to achieve defined molar ratios.

Main Results:

  • The 'one-source' standard method provides accurate, site-specific quantification of protein phosphorylation.
  • The internal standard ensures accurate measurement independent of absolute analyte concentration.
  • Successful determination of phosphorylation degrees for signaling proteins STAT5A/B and STAT6 was achieved.

Conclusions:

  • The developed method offers a robust approach for precise protein phosphorylation degree determination.
  • This technique enhances the understanding of signal transduction by accurately quantifying phosphorylation events.
  • The method is applicable to various signaling proteins, advancing proteomic analysis.