Department of Chemistry, Molecular Biosciences and the National Resource for Translational and Developmental Proteomics, Northwestern University, 2170 Campus Drive, Evanston, IL, 60208, USA.
Department of Medicine, Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
J Am Soc Mass Spectrom. 2019 Dec;30(12):2526-2534. doi: 10.1007/s13361-019-02255-x. Epub 2019 Jul 8.
Histone posttranslational modifications (PTMs) are essential for regulating chromatin and maintaining gene expression throughout cell differentiation. Despite the deep level of understanding of immunophenotypic differentiation pathways in hematopoietic cells, few studies have investigated global levels of histone PTMs required for differentiation and maintenance of these distinct cell types. Here, we describe an approach to couple fluorescence-activated cell sorting (FACS) with targeted mass spectrometry to define global "epi-proteomic" signatures for primary leukocytes. FACS was used to sort closely and distantly related leukocytes from normal human peripheral blood for quantitation of histone PTMs with a multiple reaction monitoring LC-MS/MS method measuring histone PTMs on histones H3 and H4. We validate cell sorting directly into HSO for immediate histone extraction to decrease time and number of steps after FACS to analyze histone PTMs. Relative histone PTM levels vary in T cells across healthy donors, and the majority of PTMs remain stable up to 2 days following initial blood draw. Large differences in the levels of histone PTMs are observed across the mature lymphoid and myeloid lineages, as well as between different types within the same lineage, though no differences are observed in closely related T cell subtypes. The results show a streamlined approach for quantifying global changes in histone PTMs in cell types separated by FACS that is poised for clinical deployment.
组蛋白翻译后修饰 (PTMs) 对于调节染色质和维持细胞分化过程中的基因表达至关重要。尽管人们对造血细胞免疫表型分化途径有了深入的了解，但很少有研究调查过分化和维持这些不同细胞类型所需的组蛋白 PTM 的整体水平。在这里，我们描述了一种将荧光激活细胞分选 (FACS) 与靶向质谱相结合的方法，以定义初级白细胞的全局“表观蛋白质组”特征。FACS 用于从小鼠外周血中分离密切相关和远距离相关的白细胞，并用一种测量组蛋白 H3 和 H4 上组蛋白 PTM 的多重反应监测 LC-MS/MS 方法来定量组蛋白 PTM。我们验证了直接将细胞分选到 HSO 中进行即时组蛋白提取，以减少 FACS 后分析组蛋白 PTM 的步骤和时间。健康供体的 T 细胞中组蛋白 PTM 水平存在差异，并且大多数 PTM 在初始采血后 2 天内保持稳定。在成熟的淋巴样和髓样谱系中以及同一谱系内的不同类型之间观察到组蛋白 PTM 水平的巨大差异，但在密切相关的 T 细胞亚型中未观察到差异。结果表明，这是一种通过 FACS 分离的细胞类型定量组蛋白 PTM 整体变化的简化方法，为临床应用做好了准备。
