Trelle Morten B, Salcedo-Amaya Adriana M, Cohen Adrian M, Stunnenberg Hendrik G, Jensen Ole N
Department of Biochemistry and Molecular Biology, Protein Research Group, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
J Proteome Res. 2009 Jul;8(7):3439-50. doi: 10.1021/pr9000898.
Post-translational modifications (PTMs) of histone tails play a key role in epigenetic regulation of gene expression in a range of organisms from yeast to human; however, little is known about histone proteins from the parasite that causes malaria in humans, Plasmodium falciparum. We characterized P. falciparum histone PTMs using advanced mass spectrometry driven proteomics. Acid-extracted proteins were resolved in SDS-PAGE, in-gel trypsin digested, and analyzed by reversed-phase LC-MS/MS. Through the combination of Q-TOF and LTQ-FT mass spectrometry we obtained high mass accuracy of both precursor and fragment ions, which is a prerequisite for high-confidence identifications of multisite peptide modifications. We utilize MS/MS fragment marker ions to validate the identification of histone modifications and report the m/z 143 ion as a novel MS/MS marker ion for monomethylated lysine. We identified all known P. falciparum histones and mapped 44 different modifications, providing a comprehensive view of epigenetic marks in the parasite. Interestingly, the parasite exhibits a histone modification pattern that is distinct from its human host. A general preponderance for modifications associated with a transcriptionally permissive state was observed. Additionally, a novel differentiation in the modification pattern of the two histone H2B variants (H2B and H2Bv) was observed, suggesting divergent functions of the two H2B variants in the parasite. Taken together, our results provide a first comprehensive map of histone modifications in P. falciparum and highlight the utility of tandem MS for detailed analysis of peptides containing multiple PTMs.
组蛋白尾部的翻译后修饰（PTMs）在从酵母到人类等一系列生物体的基因表达表观遗传调控中发挥着关键作用；然而，对于导致人类疟疾的疟原虫——恶性疟原虫的组蛋白却知之甚少。我们使用先进的质谱驱动蛋白质组学对恶性疟原虫组蛋白PTMs进行了表征。酸提取的蛋白质在SDS-PAGE中分离，进行胶内胰蛋白酶消化，然后通过反相LC-MS/MS进行分析。通过结合Q-TOF和LTQ-FT质谱，我们获得了前体离子和碎片离子的高质量准确度，这是高可信度鉴定多位点肽修饰的先决条件。我们利用MS/MS碎片标记离子来验证组蛋白修饰的鉴定，并报告m/z 143离子作为单甲基化赖氨酸的新型MS/MS标记离子。我们鉴定了所有已知的恶性疟原虫组蛋白，并绘制了44种不同的修饰图谱，提供了该寄生虫表观遗传标记的全面视图。有趣的是，该寄生虫呈现出与其人类宿主不同的组蛋白修饰模式。观察到与转录允许状态相关的修饰普遍占优势。此外，还观察到两种组蛋白H2B变体（H2B和H2Bv）修饰模式的新差异，表明这两种H2B变体在寄生虫中具有不同的功能。综上所述，我们的结果提供了恶性疟原虫组蛋白修饰的首张全面图谱，并突出了串联质谱在详细分析含有多个PTMs的肽段方面的实用性。
