title: Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.
abstract: Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction.
authors.affiliation: Secció de Proteòmica, Servei Central de Suport a la Investigació Experimental (SCSIE), Universitat de València, C/Dr. Moliner 50, 46100, Burjassot, València, Spain. Electronic address: mluz.valero@uv.es.
authors.forename: M Luz
authors.initials: ML
authors.lastname: Valero
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, C/Dr. Moliner 50, 46100, Burjassot, València, Spain. Electronic address: ramon.sendra@uv.es.
authors.forename: Ramon
authors.initials: R
authors.lastname: Sendra
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, C/Dr. Moliner 50, 46100, Burjassot, València, Spain. Electronic address: merce.pamblanco@uv.es.
authors.forename: Mercè
authors.initials: M
authors.lastname: Pamblanco
mesh_terms: D006657:Histones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D006657:Histones; D029701:Saccharomyces cerevisiae Proteins
keywords: Chromatin; Histones; Post-translational modifications; Proteomics; Tandem affinity purification; Yeast