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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...
Protein Kinases and Phosphatases02:54

Protein 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...
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...

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Related Experiment Video

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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
07:45

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Published on: June 6, 2022

Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression

Ronghu Wu1, Noah Dephoure, Wilhelm Haas

  • 1Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Molecular & Cellular Proteomics : MCP
|May 10, 2011
PubMed
Summary

Interpreting phosphoproteomics is challenging due to combined protein and phosphorylation changes. This study calibrates phosphopeptide ratios with protein levels, revealing 25% of changes were due to protein expression, not phosphorylation.

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Last Updated: Jun 2, 2026

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

Area of Science:

  • Proteomics
  • Yeast genetics
  • Molecular cell biology

Background:

  • Quantitative phosphoproteomics interpretation is complex, integrating both protein expression and phosphorylation changes.
  • Distinguishing true phosphorylation events from those altered by protein abundance is crucial for accurate biological insights.

Purpose of the Study:

  • To investigate the interplay between protein expression and phosphorylation changes in yeast.
  • To develop a method for calibrating phosphopeptide ratios with protein levels for accurate differential phosphorylation analysis.

Main Methods:

  • Parallel quantitative analysis of protein expression and phosphorylation in Saccharomyces cerevisiae (yeast) mutants.
  • Utilized mass spectrometry to quantify over 4100 proteins and identify 12,499 unique phosphorylation sites.
  • Developed and applied a novel method to assess protein false-discovery rate estimates and calibrate phosphopeptide ratios.

Main Results:

  • Successfully calibrated 96% of nonredundant phosphopeptide ratios using protein levels.
  • Revealed that 25% of previously identified differential phosphopeptides were actually due to changes in protein expression.
  • Uncovered independent and concerted changes in protein expression and phosphorylation, highlighting functional redundancy in the yeast mating pathway.

Conclusions:

  • Calibrating phosphopeptide data with protein expression levels is essential for accurate interpretation of phosphoproteomics studies.
  • A significant proportion of differential phosphorylation signals can be attributed to altered protein abundance.
  • This approach provides a more precise understanding of signaling pathway regulation, including roles of kinases like Kss1.