title: Identification and Quantification of Histone PTMs Using High-Resolution Mass Spectrometry.
abstract: DNA is organized into nucleosomes, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins including H2A, H2B, H3, and H4. Histones are critical regulators of many nuclear processes, including transcription, DNA damage repair, and higher order chromatin structure. Much of their function is mediated through extensive and dynamic posttranslational modification (PTM) by nuclear enzymes. Histone PTMs are thought to form a code, where combinations of PTMs are responsible for specific biological functions. Here, we present protocols to identify and quantify histone PTMs using nanoflow liquid chromatography coupled to mass spectrometry (MS). We first describe how to purify histones and prepare them for MS. We then describe three MS platforms for histone PTM analysis, including bottom-up, middle-down, and top-down approaches, and explain the relative benefits and pitfalls of each approach. We also include tips to increase the throughput of large experiments.
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: K R
authors.initials: KR
authors.lastname: Karch
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: S
authors.initials: S
authors.lastname: Sidoli
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: bgarci@mail.med.upenn.edu.
authors.forename: B A
authors.initials: BA
authors.lastname: Garcia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006657:Histones
keywords: Bottom-up; Epigenetics; Histone; Mass spectrometry; Middle-down; Posttranslational modifications; Top-down