Novel IEF Peptide Fractionation Method Reveals a Detailed Profile of N-Terminal Acetylation in Chemotherapy-Responsive and -Resistant Ovarian Cancer Cells

J Proteome Res. 2016 Nov 4;15(11):4073-4081. doi: 10.1021/acs.jproteome.6b00053. Epub 2016 Sep 19.


Although acetylation is regarded as a common protein modification, a detailed proteome-wide profile of this post-translational modification may reveal important biological insight regarding differential acetylation of individual proteins. Here we optimized a novel peptide IEF fractionation method for use prior to LC-MS/MS analysis to obtain a more in depth coverage of N-terminally acetylated proteins from complex samples. Application of the method to the analysis of the serous ovarian cancer cell line OVCAR-5 identified 344 N-terminally acetylated proteins, 12 of which are previously unreported. The protein peptidyl-prolyl cis-trans isomerase A (PPIA) was detected in both the N-terminally acetylated and unmodified forms and was further analyzed by data-independent acquisition in carboplatin-responsive parental OVCAR-5 cells and carboplatin-resistant OVCAR-5 cells. This revealed a higher ratio of unacetylated to acetylated N-terminal PPIA in the parental compared with the carboplatin-resistant OVCAR-5 cells and a 4.1-fold increase in PPIA abundance overall in the parental cells relative to carboplatin-resistant OVCAR-5 cells (P = 0.015). In summary, the novel IEF peptide fractionation method presented here is robust, reproducible, and can be applied to the profiling of N-terminally acetylated proteins. All mass spectrometry data is available as a ProteomeXchange repository (PXD003547).

Keywords: N-terminal acetylation; PPIA; carboplatin resistance; chemoresistance; ovarian cancer; peptidyl-prolyl cis−trans isomerase A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation*
  • Antineoplastic Agents / pharmacology
  • Carboplatin / pharmacology
  • Cell Line, Tumor
  • Chemical Fractionation / methods
  • Databases, Protein
  • Drug Resistance, Neoplasm*
  • Female
  • Humans
  • Mass Spectrometry
  • Ovarian Neoplasms / drug therapy*
  • Peptide Fragments / analysis*
  • Protein Processing, Post-Translational
  • Proteomics / methods


  • Antineoplastic Agents
  • Peptide Fragments
  • Carboplatin