Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction

Mol Cell Proteomics. 2016 Aug;15(8):2576-93. doi: 10.1074/mcp.M116.058420. Epub 2016 May 16.


Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphatases / antagonists & inhibitors
  • Binding Sites
  • Cell Cycle Proteins / antagonists & inhibitors
  • Chromatography, Liquid
  • HCT116 Cells
  • Homeostasis / drug effects
  • Humans
  • Proteasome Inhibitors / pharmacology*
  • Protein Interaction Maps
  • Proteome / chemistry
  • Proteome / drug effects
  • Proteome / metabolism*
  • Proteomics / methods*
  • Tandem Mass Spectrometry
  • Ubiquitins / metabolism*
  • Valosin Containing Protein


  • Cell Cycle Proteins
  • Proteasome Inhibitors
  • Proteome
  • Ubiquitins
  • Adenosine Triphosphatases
  • VCP protein, human
  • Valosin Containing Protein