Isolation of Proteasome-Trapped Peptides (PTPs) for Degradome Analysis

Indrajit Sahu, Manisha Priyadarsini Sahoo, Oded Kleifeld, Michael H. Glickman

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


Analyzing intracellular peptides generated by proteasomes is highly informative to understand the spatiotemporal regulation of protein homeostasis. A large portion of eukaryotic proteins is proteolyzed within the 20S core particle of the 26S holoenzyme, where proteins are cleaved into peptides of varying lengths. A small percentage of these peptides are presented to the immune system as a representation of the proteome content of the cell. Therefore, understanding the rules that govern proteolytic specificity and product diversity is of relevance not only to biochemistry and proteostasis but also to physiology and immunology. One of the greatest challenges is to separate such proteasome-generated peptides from the total intracellular peptidome due to the susceptibility of short unstructured peptides to myriad proteases and peptidases that are activated upon cell lysis. Here, we describe a simple and rapid method to isolate peptides that are closely associated with proteasomes or trapped inside the core particle of proteasomes in eukaryotic cells. This approach termed PTPs, for proteasome-trapped peptides, requires a limited number of cells as starting materials compared to other published methods yet still provides sufficient yields for mass spectrometry-based proteomic analysis. A single sample obtained from cultured mammalian cells allowed the identification of 1000–2000 different PTPs following LC-MS analysis with high-resolution mass spectrometer.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Number of pages13
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • 20S CP
  • 26S holoenzyme
  • Degradome
  • Intracellular peptides
  • PTPs
  • Peptidome
  • Peptidomics
  • Proteasome
  • Proteasome-trapped peptides
  • Proteasome-trapped peptides
  • Proteomics
  • Ubiquitin
  • Ultracentrifuge

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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