Berryhill Christine A, Evans Taylor N, Doud Emma H, Smith-Kinnaman Whitney R, Hanquier Jocelyne N, Mosley Amber L, Cornett Evan M
J Proteome Res. 2025 Feb 7;24(2):550-561. doi: 10.1021/acs.jproteome.4c00685. Epub 2025 Jan 8.
Growing evidence shows that lysine methylation is a widespread protein post-translational modification (PTM) that regulates protein function on histone and nonhistone proteins. Numerous studies have demonstrated that the dysregulation of lysine methylation mediators contributes to cancer growth and chemotherapeutic resistance. While changes in histone methylation are well-documented with extensive analytical techniques available, there is a lack of high-throughput methods to reproducibly quantify changes in the abundances of the mediators of lysine methylation and nonhistone lysine methylation (Kme) simultaneously across multiple samples. Recent studies by our group and others have demonstrated that antibody enrichment is not required to detect lysine methylation, prompting us to investigate the use of tandem mass tag (TMT) labeling for global Kme quantification without antibody enrichment in four different breast cancer cell lines (MCF-7, MDA-MB-231, HCC1806, and MCF10A). To improve the quantification of KDMs, we incorporated a lysine demethylase (KDM) isobaric trigger channel, which enabled 96% of all KDMs to be quantified while simultaneously quantifying 326 Kme sites. Overall, 142 differentially abundant Kme sites and eight differentially abundant KDMs were identified among the four cell lines, revealing cell line-specific patterning.
越来越多的证据表明，赖氨酸甲基化是一种广泛存在的蛋白质翻译后修饰（PTM），可调节组蛋白和非组蛋白的蛋白质功能。大量研究表明，赖氨酸甲基化介质的失调会促进癌症生长和化疗耐药性。虽然通过现有的广泛分析技术，组蛋白甲基化的变化已有充分记录，但缺乏高通量方法来在多个样本中同时可重复地定量赖氨酸甲基化介质和非组蛋白赖氨酸甲基化（Kme）丰度的变化。我们团队和其他团队最近的研究表明，检测赖氨酸甲基化不需要抗体富集，这促使我们研究使用串联质量标签（TMT）标记在四种不同的乳腺癌细胞系（MCF-7、MDA-MB-231、HCC1806和MCF10A）中进行无需抗体富集的全局Kme定量。为了改进对赖氨酸去甲基化酶（KDM）的定量，我们引入了一个赖氨酸去甲基化酶等压触发通道，该通道能够在同时定量326个Kme位点的情况下对所有KDM中的96%进行定量。总体而言，在这四种细胞系中鉴定出了142个差异丰度的Kme位点和8个差异丰度的KDM，揭示了细胞系特异性模式。
