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

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).