title: Quantification of histone modifications by parallel-reaction monitoring: a method validation.
abstract: Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Anal. Chem. 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knockout mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with ascorbic acid phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by 6 days of treatment with vitamin C.
authors.forename: James L
authors.initials: JL
authors.lastname: Sowers
authors.forename: Barsam
authors.initials: B
authors.lastname: Mirfattah
authors.forename: Pei
authors.initials: P
authors.lastname: Xu
authors.forename: Hui
authors.initials: H
authors.lastname: Tang
authors.affiliation: Institute of Biosciences and Technology, Texas A&M Health Sciences Center , Houston, Texas 77030, United States.
authors.forename: In Young
authors.initials: IY
authors.lastname: Park
authors.affiliation: Institute of Biosciences and Technology, Texas A&M Health Sciences Center , Houston, Texas 77030, United States.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Walker
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.forename: Fernanda
authors.initials: F
authors.lastname: Laezza
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Sowers
authors.forename: Kangling
authors.initials: K
authors.lastname: Zhang
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D044127:Epigenesis, Genetic; D042421:Histone Code; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D006657:Histones / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008745:Methylation; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D058953:Neural Stem Cells / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D006657:Histones; D011495:Histone-Lysine N-Methyltransferase; C587921:SETD2 protein, mouse; D014357:Trypsin