Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
Viruses. 2020 Dec 8;12(12):1409. doi: 10.3390/v12121409.
T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.
T 细胞的功能取决于转录网络，这些网络受表观遗传编程调控，通过组蛋白和 DNA 的翻译后修饰（PTMs）进行调控。自下而上的质谱（MS）可以鉴定组蛋白 PTM，而通过 MS 进行的完整蛋白质分析可以检测到通过自下而上方法错过的物种。我们使用了一种新颖的方法，即在线二维液相色谱-串联 MS 与高分辨率反相液相色谱（RPLC）相结合，在先导离子上交替进行电子转移解离（ETD）和碰撞诱导解离（CID），以最大限度地对独特修饰的物种进行碎片化。第一个在线 RPLC 分离按家族对组蛋白进行分类，然后 RPLC 或弱阳离子交换亲水相互作用液相色谱（WCX-HILIC）对大量覆盖 PTM 的物质进行分离。通过将蛋白质质量与预测的理论质量相匹配来分配暂定鉴定，然后通过串联 MS 进行验证。我们使用这种创新的方法进行组蛋白完整蛋白 PTM 图谱分析（HiPTMap），以鉴定和定量从体内流感感染后的 CD8 T 细胞中纯化的蛋白质。流感感染后，PTMs 的激活发生了显著改变，T 细胞迁移到感染部位时组蛋白图谱发生了变化，而对二次感染有反应的 T 细胞具有明显更多的转录增强修饰。因此，HiPTMap 鉴定和定量了蛋白质，并确定了 CD8 T 细胞组蛋白 PTM 在感染过程中的变化。
