Application of mass spectrometry to the identification and quantification of histone post‐translational modifications

Freitas, Michael A.; Sklenar, Amy R.; Parthun, Mark R.

doi:10.1002/jcb.20106

Cited by 130 publications

(98 citation statements)

References 51 publications

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“…For desalting, 5-10 l of peptide mixtures were absorbed on a POROS R3 column, washed with 0.2% formic acid, and desorbed sequentially with 2 l of 10, 20, 30, and 50% methanol in 0.2% formic acid. Peptides H3-(1-19), H3-(9 -17), H3- (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), and H2B-(1-35) were eluted at 10% methanol. Peptides H2A-(1-41) (for the first and second HPLC peaks) and peptides H3-(73-83) were eluted at 50% methanol after 10, 20, and 30% step elutions.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry

Bonenfant

Towbin

Coulot

et al. 2007

Molecular & Cellular Proteomics

125

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The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G 1 , S, and G 2 /M. For all core histones, the modifications in the G 1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G 2 /M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G 2 /M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G 2 /M, H3 Lys 27 and Lys 36were decreased, whereas H4 Lys 20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.

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Section: Methodsmentioning

confidence: 99%

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry

Bonenfant

Towbin

Coulot

et al. 2007

Molecular & Cellular Proteomics

125

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“…Their characterization is complex due to the existence of sequence variants and their high degree of post-translational modifications (PTMs). Alterations in histone PTMs have been documented in many types of cancer and may in fact play an important role in tumorogenesis [2]. The National Human Genome Research Institute (NHGRI) maintains a histone database (http://research.nhgri.nih.gov/histones/) that list 21 variants of human H2A, 14 of H2B, and at least three variants of H3 [3], in addition to proteins similar to core histones or histone-like.…”

Section: Introductionmentioning

confidence: 99%

Liquid chromatography mass spectrometry profiling of histones

Jacob

Amunugama

et al. 2007

Journal of Chromatography B

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Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RP-LC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.

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“…[112][113][114][115][116] Thus far, a myriad of lysine and arginine residues in core histones are found to be methylated ( Figure 5). As a matter of fact, the majority of these methylation sites were discovered by MS methods.…”

Section: Analysis Of Histone Methylation By Mass Spectrometrymentioning

confidence: 99%

Chemical and biochemical approaches in the study of histone methylation and demethylation

Luo

Wang

et al. 2010

Med. Res. Rev.

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Histone methylation represents one of the most critical epigenetic events in DNA function regulation in eukaryotic organisms. Classic molecular biology and genetics tools provide significant knowledge about mechanisms and physiological roles of histone methyltransferases and demethylases in various cellular processes. In addition to this stream line, development and application of chemistry and chemistry-related techniques are increasingly involved in biological study, and provide information otherwise difficulty to obtain by standard molecular biology methods. Herein, we review recent achievements and progress in developing and applying chemical and biochemical approaches in the study of histone methylation, including chromatin immunoprecipitation (ChIP), chemical ligation, mass spectrometry (MS), biochemical assays, and inhibitor development. These technological advances allow histone methylation to be studied from genome-wide level to molecular and atomic levels. With ChIP technology, information can be obtained about precise mapping of histone methylation patterns at specific promoters, genes or other genomic regions. MS is particularly useful in detecting and analyzing methylation marks in histone and nonhistone protein substrates. Chemical approaches that permit site-specific incorporation of methyl groups into histone proteins greatly facilitate the investigation of the biological impacts of methylation at individual modification sites. Discovery and design of selective organic inhibitors of histone methyltransferases and demethylases provide chemical probes to interrogate methylation-mediated cellular pathways. Overall, these chemistry-related technological advances have greatly improved our understanding of the biological functions of histone methylation in normal physiology and diseased states, and also are of great potential to translate basic epigenetics research into diagnostic and therapeutic application in the clinic.

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Application of mass spectrometry to the identification and quantification of histone post‐translational modifications

Cited by 130 publications

References 51 publications

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry

Liquid chromatography mass spectrometry profiling of histones

Chemical and biochemical approaches in the study of histone methylation and demethylation

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