Kim Sun-Yee, Sim Choon Kiat, Zhang Qiongyi, Tang Hui, Brunmeir Reinhard, Pan Hong, Karnani Neerja, Han Weiping, Zhang Kangling, Xu Feng
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America.
PLoS One. 2016 Sep 8;11(9):e0162528. doi: 10.1371/journal.pone.0162528. eCollection 2016.
Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.
赖氨酸乙酰化是细胞信号传导中一种重要的翻译后修饰。在乙酰化蛋白质组研究中，高质量的泛乙酰赖氨酸抗体是成功富集乙酰化肽段以供后续质谱分析的关键。在此，我们展示了一种使用合成的乙酰化肽段随机文库作为抗原生成多克隆泛乙酰赖氨酸抗体的替代方法。我们的抗体通过酶联免疫吸附测定（ELISA）和斑点印迹法检测，对乙酰赖氨酸肽段/蛋白质具有特异性。将我们的五种抗体混合后，对乙酰赖氨酸肽段具有广泛的反应性，在肽段覆盖范围方面可补充一种商业抗体。我们的抗体混合物所结合肽段的共有序列与商业抗体的略有不同。最后，我们的抗体在一个概念验证实验中用于分析人胚肾293（HEK293）细胞的乙酰化蛋白质组。我们总共从416种蛋白质中鉴定出1557个乙酰化肽段。因此，我们证明了我们的抗体非常适合用于乙酰化蛋白质组研究，并且可以补充现有的商业抗体。
