Highly Efficient Separation of Methylated Peptides Utilizing Selective Complexation between Lysine and 18-Crown‑6
Highly Efficient Separation of Methylated Peptides Utilizing Selective Complexation between Lysine and 18-Crown‑6
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利用赖氨酸和 18-Crown 之间的选择性络合高效分离甲基化肽 6
DOI：
10.1021/acs.analchem.0c04158
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发表时间：
2020
影响因子：
7.4
通讯作者：
Xinmiao Liang
中科院分区：
文献类型：
--
作者：
Qianying Sheng;Cunli Wang;Xiaopei Li;Hongqiang Qin;Mingliang Ye;Yuting Xiong;Xue Wang;Xiuling Li;Minbo Lan;Junyan Li;Yanxiong Ke;Guangyan Qing;Xinmiao Liang
蛋白质甲基化是最常见、最重要的翻译后修饰之一，在表观遗传调控、信号转导和染色质代谢中起着至关重要的作用。然而，由于甲基化形式的多样性，甲基化位点与未修饰位点之间的微小差异，以及极低的丰度，从生物样品中捕获和分离甲基化肽是非常有挑战性的。在这里，我们介绍了一种简单高效的分离甲基化和非甲基化肽的方法，使用18-冠-6作为高效液相色谱的流动相添加剂。赖氨酸和18-冠-6之间的选择性络合显著增加了肽在C18固定相上的保留，导致赖氨酸甲基化和非甲基化肽之间的良好基线分离。核磁共振滴定、生物层干涉技术和量子化学计算验证了一种可能的结合机制。通过建立一种简单的富集方法，实现了良好的选择性，并成功地从含有10倍牛血清白蛋白胰蛋白酶消化的复杂肽样品中分离出4个具有显著提高的信噪比（S/N）的甲基化肽。选择rLys N作为消化组蛋白的酶，根据我们的富集方法可以很好地识别组蛋白中的甲基化信息。本研究将为翻译后修饰蛋白质组学中赖氨酸甲基化肽的选择性富集开辟新的途径。
Protein methylation is one of the most common and important post-translational modifications, and it plays vital roles in epigenetic regulation, signal transduction, and chromatin metabolism. However, due to the diversity of methylation forms, slight difference between methylated sites and nonmodified ones, and ultralow abundance, it is extraordinarily challenging to capture and separate methylated peptides from biological samples. Here, we introduce a simple and highly efficient method to separate methylated and nonmethylated peptides using 18-crown-6 as a mobile phase additive in high-performance liquid chromatography. Selective complexation between lysine and 18-crown-6 remarkably increases the retention of the peptides on a C18 stationary phase, leading to an excellent baseline separation between the lysine methylated and nonmethylated peptides. A possible binding mechanism is verified by nuclear magnetic resonance titration, biolayer interferometry technology, and quantum chemistry calculation. Through establishment of a simple enrichment methodology, a good selectivity is achieved and four methylated peptides with greatly improved signal-to-noise (S/N) ratios are successfully separated from a complex peptide sample containing 10-fold bovine serum albumin tryptic digests. By selecting rLys N as an enzyme to digest histone, methylation information in the histone could be well identified based on our enrichment method. This study will open an avenue and provide a novel insight for selective enrichment of lysine methylated peptides in post-translational modification proteomics.