Weiland Florian, Arentz Georgia, Klingler-Hoffmann Manuela, McCarthy Peter, Lokman Noor A, Kaur Gurjeet, Oehler Martin K, Hoffmann Peter
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.
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).
尽管乙酰化被视为一种常见的蛋白质修饰，但这种翻译后修饰的详细全蛋白质组图谱可能揭示有关单个蛋白质差异乙酰化的重要生物学见解。在这里，我们优化了一种新型肽IEF分级分离方法，以便在LC-MS/MS分析之前使用，以更深入地覆盖复杂样品中N端乙酰化的蛋白质。将该方法应用于浆液性卵巢癌细胞系OVCAR-5的分析，鉴定出344种N端乙酰化蛋白质，其中12种以前未被报道。在N端乙酰化和未修饰形式中均检测到蛋白质肽基脯氨酰顺反异构酶A（PPIA），并在对卡铂敏感的亲本OVCAR-5细胞和对卡铂耐药的OVCAR-5细胞中通过数据非依赖采集进一步分析。这表明，与对卡铂耐药的OVCAR-5细胞相比，亲本细胞中未乙酰化与乙酰化的N端PPIA比例更高，并且亲本细胞中PPIA的总体丰度相对于对卡铂耐药的OVCAR-5细胞增加了4.1倍（P = 0.015）。总之，本文提出的新型IEF肽分级分离方法稳健、可重复，可应用于N端乙酰化蛋白质的分析。所有质谱数据可在ProteomeXchange储存库（PXD003547）中获取。
