Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin

Donald S. Kirkpatrick, Stephen F. Weldon, George Tsaprailis, Daniel C. Liebler, A. Jay Gandolfi

Research output: Contribution to journalArticle

76 Scopus citations

Abstract

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST®. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 μM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.

Original languageEnglish (US)
Pages (from-to)2104-2111
Number of pages8
JournalProteomics
Volume5
Issue number8
DOIs
StatePublished - May 2005

Keywords

  • Arsenic
  • Green fluorescent protein
  • HEK293
  • Mass spectrometry
  • Nickel purification
  • Ubiquitin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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