Histones posttranslational modification represent an epigenetic mechanism that regulate gene expression and other cellular processes. Quantitative mass spectrometry used for the absolute quantification of such modifications provides further insight into cellular responses to extracellular insults such as infections or toxins. Methamphetamine (Meth), a drug of abuse, is affecting the overall function of the immune system. In this report, we developed, validated and applied a targeted, MS-based quantification assay to measure changes in histone H3 lysine 14 acetylation (H3K14Ac) during exposure of human primary macrophages to HIV-1 infection and/or Meth. The quantification assay was developed and validated to determine H3K14Ac stoichiometry in histones that were isolated from the nuclei of control (CIC) and exposed to Meth before (CIM) or/and after (MIM) HIV-infection human monocyte-derived macrophages (hMDM) of six donors. It was based on LC–MS/MS measurement using multiple reaction monitoring (MRM) acquisition of the unmodified and acetylated form of lysine K14 of histone H3 9KSTGGKAPR17 peptides and the corresponding stable isotope labeled (SIL) heavy peptide standards of the same sequences. The histone samples were propionylated (Poy) pre- and post- trypsin digestion so that the sequences of the monitored peptides were: K[Poy]STGGK[1Ac]APR, K[Poy]STGGK[1Ac]APR-heavy, K[Poy]STGGK[Poy]APR and K[Poy]STGGK[Poy]APR-heavy. The absolute amounts of the acetylated and unmodified peptides were determined by comparing to the abundances of their SIL standards, that were added to the samples in the known concentrations, and, then used for calculation of H3K14Ac stoichiometry in CIC, CIM and MIM hMDM. The assay was characterized by LLOD of 0.106 fmol/µL and 0.204 fmol/µL for unmodified and acetylated H3 9KSTGGKAPR17 peptides, respectively. The LLOQ was 0.5 fmol/µL and the linear range of the assay was from 0.5 to 2500 fmol/µL. The absolute abundances of the quantified peptides varied between the donors and conditions, and so did the H3K14Ac stoichiometry. This was rather attributed to the samples nature itself, as the variability of their triplicate measurements was low. The developed LC–MS/MS assay enabled absolute quantification of H3K14Ac in exposed to Meth HIV-infected hMDM. It can be further applied determination of this PTM stoichiometry in other studies on human primary macrophages.

中文翻译：

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基于 MS 的绝对靶向定量蛋白质组学方法测定人单核细胞来源的巨噬细胞中组蛋白 H3 赖氨酸 14 乙酰化化学计量的变化：HIV 感染和甲基苯丙胺暴露

组蛋白翻译后修饰代表了调节基因表达和其他细胞过程的表观遗传机制。用于绝对定量此类修饰的定量质谱法可以进一步了解细胞对细胞外损伤（例如感染或毒素）的反应。甲基苯丙胺（Meth）是一种滥用药物，它正在影响免疫系统的整体功能。在本报告中，我们开发、验证并应用了一种基于 MS 的靶向定量测定，以测量人类原代巨噬细胞暴露于 HIV-1 感染和/或冰毒期间组蛋白 H3 赖氨酸 14 乙酰化 (H3K14Ac) 的变化。开发并验证了定量测定，以确定组蛋白中的 H3K14Ac 化学计量，这些组蛋白从对照细胞核 (CIC) 中分离出来，并在 HIV 感染人类单核细胞来源的巨噬细胞 (hMDM) 之前 (CIM) 或/和之后 (MIM) 暴露于 Meth六位捐助者。它基于 LC-MS/MS 测量，使用多反应监测 (MRM) 采集组蛋白 H3 9KSTGGKAPR17 肽的赖氨酸 K14 的未修饰和乙酰化形式以及相同序列的相应稳定同位素标记 (SIL) 重肽标准品。组蛋白样品在胰蛋白酶消化前和消化后进行丙酰化 (Poy)，以便监测的肽序列为：K[Poy]STGGK[1Ac]APR、K[Poy]STGGK[1Ac]APR-heavy、K[Poy]STGGK[1Ac]APR-heavy、K[Poy]STGGK[1Ac]APR ]STGGK[Poy]APR 和 K[Poy]STGGK[Poy]APR 重。通过与以已知浓度添加到样品中的 SIL 标准品的丰度进行比较，确定乙酰化和未修饰肽的绝对量，然后用于计算 CIC、CIM 和 MIM hMDM 中的 H3K14Ac 化学计量。该测定的特征是未修饰和乙酰化的 H3 9KSTGGKAPR17 肽的 LLOD 分别为 0.106 fmol/μL 和 0.204 fmol/μL。LLOQ 为 0.5 fmol/μL，测定的线性范围为 0.5 至 2500 fmol/μL。定量肽的绝对丰度因供体和条件的不同而不同，H3K14Ac 化学计量也不同。这很大程度上归因于样品本身的性质，因为三次重复测量的变异性很低。开发的 LC-MS/MS 检测能够对暴露于 Meth HIV 感染的 hMDM 的 H3K14Ac 进行绝对定量。它可以进一步应用于人类原代巨噬细胞的其他研究中该 PTM 化学计量的测定。