Change of histone H3 lysine 14 acetylation stoichiometry in human monocyte derived macrophages as determined by MS-based absolute targeted quantitative proteomic approach: HIV infection and methamphetamine exposure

• Published in: Clinical proteomics 2023-10, Vol.20 (1), p.48-48, Article 48
• Main Authors: Macur, Katarzyna, Schissel, Andrew, Yu, Fang, Lei, Shulei, Morsey, Brenda, Fox, Howard S, Ciborowski, Pawel
• Format: Article
• Language: English
• Subjects: Acetylation; Cell culture; DNA binding proteins; Drug abuse; Epigenetic inheritance; Epigenetics; Gene expression; Health aspects; Histone H3; Histones; HIV; HIV (Viruses); HIV infection; Human immunodeficiency virus; Immune system; Infections; Liquid chromatography; Lysine; Macrophages; Mass spectrometry; Mass spectroscopy; Methamphetamine; Monocytes; Peptides; Post-translational modification; Proteomics; Scientific imaging; Stable isotopes; Stoichiometry; Toxins; Trypsin; Viral infections
• ISSN: 1542-6416; 1559-0275
• DOI: 10.1186/s12014-023-09438-5

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 KSTGGKAPR 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 KSTGGKAPR 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.