School of Life Sciences, The Chinese University of Hong Kong, Sha Tin, Hong Kong 999077, P. R. China.
State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 999077, P. R. China.
J Proteome Res. 2021 Sep 3;20(9):4331-4345. doi: 10.1021/acs.jproteome.1c00308. Epub 2021 Jul 30.
The heterogeneity of histone H3 proteoforms makes histone H3 top-down analysis challenging. To enhance the detection coverage of the proteoforms, performing liquid chromatography (LC) front-end to mass spectrometry (MS) detection is recommended. Here, using optimized electron-transfer/high-energy collision dissociation (EThcD) parameters, we have conducted a proteoform-spectrum match (PrSM)-level side-by-side comparison of reversed-phase LC-MS (RPLC-MS), "dual-gradient" weak cation-exchange/hydrophilic interaction LC-MS (dual-gradient WCX/HILIC-MS), and "organic-rich" WCX/HILIC-MS on the top-down analyses of H3.1, H3.2, and H4 proteins extracted from a HeLa cell culture. While both dual-gradient WCX/HILIC and organic-rich WCX/HILIC could resolve intact H3 and H4 proteoforms by the number of acetylations, the organic-rich method could enhance the separations of different trimethyl/acetyl near-isobaric H3 proteoforms. In comparison with RPLC-MS, both of the WCX/HILIC-MS methods enhanced the qualities of the H3 PrSMs and remarkably improved the range, reproducibility, and confidence in the identifications of H3 proteoforms.
组蛋白 H3 蛋白变体的异质性使得 H3 从头分析具有挑战性。为了提高蛋白变体的检测覆盖率，建议在液质联用（LC-MS）检测之前进行液相色谱（LC）前端处理。在这里，我们使用优化的电子转移/高能碰撞解离（EThcD）参数，在从头分析从 HeLa 细胞培养物中提取的 H3.1、H3.2 和 H4 蛋白时，对反相 LC-MS（RPLC-MS）、“双梯度”弱阳离子交换/亲水相互作用 LC-MS（dual-gradient WCX/HILIC-MS）和“富含有机相”WCX/HILIC-MS 进行了基于蛋白谱匹配（PrSM）水平的平行比较。虽然双梯度 WCX/HILIC 和富含有机相 WCX/HILIC 都可以通过乙酰化数量来解析完整的 H3 和 H4 蛋白变体，但富含有机相的方法可以增强不同三甲基/乙酰化近等电点 H3 蛋白变体的分离度。与 RPLC-MS 相比，WCX/HILIC-MS 方法都提高了 H3 PrSM 的质量，并显著提高了 H3 蛋白变体鉴定的范围、重现性和置信度。
