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J Proteome Res. 2016 Nov 4;15(11):4073-4081. Epub 2016 Sep 19.

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

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Adelaide Proteomics Centre, School of Biological Sciences, The University of Adelaide , Adelaide, South Australia 5005, Australia.
The Institute for Photonics & Advanced Sensing (IPAS), The University of Adelaide , Adelaide, South Australia 5005, Australia.
Department of Human Immunology, Centre for Cancer Biology, University of South Australia , Adelaide, South Australia 5000, Australia.
Robinson Institute, Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, University of Adelaide , Adelaide, South Australia 5005, Australia.
Institute for Research in Molecular Medicine, Universiti Sains Malaysia , 11800 Minden, Pulau Pinang, Malaysia.
Department of Gynaecological Oncology, Royal Adelaide Hospital , Adelaide, South Australia 5005, Australia.


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).


N-terminal acetylation; PPIA; carboplatin resistance; chemoresistance; ovarian cancer; peptidyl-prolyl cis‚ąítrans isomerase A

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