title: Profiling post-translational modifications of histones in human monocyte-derived macrophages.
abstract: BACKGROUND
  Histones 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.
  
  
  METHODS
  To 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5880 USA ; Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7 Street, 87-100 Torun, Poland.
authors.forename: Pawel
authors.initials: P
authors.lastname: Olszowy
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5880 USA.
authors.forename: Maire Rose
authors.initials: MR
authors.lastname: Donnelly
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5880 USA.
authors.forename: Chanho
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5880 USA.
authors.forename: Pawel
authors.initials: P
authors.lastname: Ciborowski
keywords: Histones; Innate immunity; Macrophage; Mass spectrometry; Post-translational modification; Proteomics