Quantification of H3K27 methylation using heavy peptides as standards

To demonstrate the usability of our SIL peptides (JPT GmbH)on an MS application, 4 standard peptides were synthesized at 95% purity and each spiked at an amount 133 fmol into protein extracts of wild type HEK cells and EZH2-knockout cells, respectively. The spiked peptides in this experiment are histone H3 K27-containing heavy-arginine versions of the peptide ARKSAPATGGVKKPH-R, where K27 was synthetically modified to carry either no methylation, or one or two or three (me3) methyl groups, respectively. Previously to trypsination, the HEK samples were spiked with the 4 SIL peptides. Because the standards have 10 Da greater mass given by the heavy R*, they can be distinguished from their light natural versions in the MS1 spectra. Furthermore, the standards were each spiked at a concentration of 133 fmol and thus, their MS intensities were used to normalize the intensities of the endogenous peptides and quantify them in absolute terms.

为验证本研究所用SIL肽（SIL peptides，JPT GmbH公司）在质谱（MS，Mass Spectrometry）分析中的应用可行性，我们合成了4种纯度达95%的标准肽段，并分别以133飞摩尔（fmol）的量添加至野生型HEK细胞与EZH2基因敲除（EZH2-knockout）细胞的蛋白提取物中。本实验所用的标准肽段为组蛋白H3来源的、带有重精氨酸标记的ARKSAPATGGVKKPH-R肽段，其K27位点经人工合成修饰，分别未引入甲基化修饰、或携带单甲基、双甲基及三甲基（me3）修饰。在进行胰蛋白酶酶解（trypsination）前，我们已向HEK细胞样品中加入上述4种SIL标准肽段。由于这些标准肽段带有重同位素标记的精氨酸（R*），其质量较天然轻肽段大10 Da，因此可在质谱一级谱图（MS1 spectra）中与内源天然肽段实现有效区分。此外，所有标准肽段均以133 fmol的固定浓度添加，因此可通过其质谱信号强度对内源肽段的信号强度进行归一化处理，最终实现内源肽段的绝对定量。