title: Characterization of Individual Histone Posttranslational Modifications and Their Combinatorial Patterns by Mass Spectrometry-Based Proteomics Strategies.
abstract: Histone posttranslational modifications (PTMs) play an essential role in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. Such PTMs are mostly localized on histone N-terminal tails where, as single units or in a combinatorial manner, they influence chromatin reader protein binding and fine-tune the abovementioned activities. Mass spectrometry (MS) is currently the most adopted strategy to characterize proteins and protein PTMs. We hereby describe the protocols to identify and quantify histone PTMs and their patterns using either bottom-up or middle-down proteomics. In the bottom-up strategy we obtain 5-20 aa peptides by derivatization with propionylation followed by trypsin digestion. The newly generated N-termini of histone peptides can be further derivatized with light or isotopically heavy propionyl groups to increase chromatographic retention and allow multiplexed analyses. Moreover, we describe how to perform derivatization and trypsin digestion of histones loaded into a gel, which is usually the final step of immunoprecipitation experiments. In the middle-down strategy we obtain intact histone tails of 50-60 aa by digestion with the enzyme GluC. This allows characterization of combinatorial histone PTMs on N-terminal tails.
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
authors.forename: Simone
authors.initials: S
authors.lastname: Sidoli
authors.affiliation: Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. bgarci@mail.med.upenn.edu.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D006657:Histones
keywords: Bottom-up; Histones; Mass spectrometry; Middle-down; Proteomics