Abstract

BackgroundHistones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms.MethodsTo profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers.ResultsWe provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages.ConclusionsThis study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0080-7) contains supplementary material, which is available to authorized users.

Highlights

  • Post-translational modifications (PTM) of histones comprise the exceptionally complex system of epigenetic regulation (“histone code”) of cellular functions

  • We present the first comprehensive approach in profiling PTMs of histones isolated from human monocyte-derived macrophages (MDM) obtained from healthy individuals

  • Sample preparation Primary human monocytes were obtained by leukophoresis from donors who were seronegative for HIV-1, HIV2, and hepatitis B virus

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Summary

Introduction

Post-translational modifications (PTM) of histones comprise the exceptionally complex system of epigenetic regulation (“histone code”) of cellular functions. The mechanisms of how microbes evade immune-surveillance as well as the effect of other factors such as exposure to toxins, illicit and other drugs etc., which contribute to a wide range of impairments of the innate immune system, are still far from being understood Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. It becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms

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