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Related Concept Videos

Mass Spectrometry: Complex Analysis01:21

Mass Spectrometry: Complex Analysis

Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...

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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
11:54

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Published on: March 23, 2020

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals.

Lilian Phu1, Anita Izrael-Tomasevic, Marissa L Matsumoto

  • 1Departments of Protein Chemistry, Genentech, Inc, South San Francisco, California 94080, USA.

Molecular & Cellular Proteomics : MCP
|November 5, 2010
PubMed
Summary

This study presents a comprehensive mass spectrometry platform to quantify ubiquitin (Ub) signals on proteins. The advanced methods reveal that cellular substrates are modified by mixed ubiquitin linkages, including K48, K63, and K11.

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Last Updated: Jun 7, 2026

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
11:54

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Published on: March 23, 2020

Ubiquitin Chain Analysis by Parallel Reaction Monitoring
08:33

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations
10:26

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

Published on: November 7, 2019

Area of Science:

  • Biochemistry
  • Proteomics
  • Cell Biology

Background:

  • Ubiquitinated substrates are recruited to cellular complexes via ubiquitin (Ub) signals and Ub receptor proteins.
  • Understanding the Ub system in vivo requires methods to determine Ub signal composition on substrates.
  • Mass spectrometry is a key tool for characterizing diverse Ub forms.

Purpose of the Study:

  • To establish a comprehensive platform for characterizing ubiquitin (Ub) signals.
  • To quantify unbranched peptides and branched -GG signature peptides generated from Ub signals.
  • To extend Ub signal characterization to include N-terminal ubiquitin, linear polyUb chains, and specific lysine linkages (K33, K48).

Main Methods:

  • Utilized the Ubiquitin-AQUA approach with synthetic, isotopically labeled internal standard peptides.
  • Analyzed peptides using selected reaction monitoring (QTRAP) or narrow window extracted ion chromatograms (LTQ-Orbitrap).
  • Integrated mass spectrometry with polyubiquitin linkage-specific antibodies (K48, K63) to analyze mixed linkage ubiquitination in cells.

Main Results:

  • Developed a comprehensive platform for Ub signal characterization, accounting for various Ub forms and digestion products.
  • Quantified total Ub in samples from multiple loci, minimizing confounding factors.
  • Demonstrated that polyubiquitinated substrates from cultured mammalian cells exhibit mixed ubiquitin linkages (K48, K63, K11).

Conclusions:

  • The established platform enables robust characterization of Ub signals in complex biological samples.
  • Combined mass spectrometry and antibody approaches reveal the prevalence of mixed ubiquitin chain linkages on cellular substrates.
  • This work advances the functional understanding of the ubiquitin system in vivo.