Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications.

The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers. Within the nucleosome, histones are decorated with post-translational modifications. Previous studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that old tetramers serve as a template for the modification of newly synthesized tetramers.

Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications.

Background: Histone isoforms and their post-translational modifications (PTMs) play an important role in the control of many chromatin-related processes including transcription and DNA damage. Variants of histones H2A and H3 have been studied in depth and have been found to have distinct functions. Although 13 somatic histone H2B isoforms have been identified by various biochemical and mass spectrometric (MS) approaches, the distinct roles of these isoforms within human cells are as yet unknown.

Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis.

MS-based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization.

Mass spectrometric analysis of histone proteoforms.

Histones play important roles in chromatin, in the forms of various posttranslational modifications (PTMs) and sequence variants, which are called histone proteoforms. Investigating modifications and variants is an ongoing challenge. Previous methods are based on antibodies, and because they usually detect only one modification at a time, they are not suitable for studying the various combinations of modifications on histones.

Proteomic characterization of novel histone post-translational modifications.

Histone post-translational modifications (PTMs) have been linked to a variety of biological processes and disease states, thus making their characterization a critical field of study. In the last 5 years, a number of novel sites and types of modifications have been discovered, greatly expanding the histone code. Mass spectrometric methods are essential for finding and validating histone PTMs.

Large-scale global identification of protein lysine methylation in vivo.

Lysine methylation mediated by methyltransferase enzymes is present on multiple proteins throughout the cell; however, methods to uncover and characterize global protein lysine methylation patterns do not readily exist. Here we developed pan-specific methyl lysine antibodies that we utilized in immunoprecipitation experiments coupled with mass spectrometry to yield one of the first large-scale surveys of protein lysine methylation in vivo. In total, 552 different lysine methylation sites were determined, making this one of the most comprehensive global studies published to date.

Revealing histone variant induced changes via quantitative proteomics.

Histone variants are isoforms of linker and core histone proteins that differ in their amino acid sequences. These variants have distinct genomic locations and posttranslational modifications, thus increasing the complexity of the chromatin architecture. Biological studies of histone variants indicate that they play a role in many processes including transcription, DNA damage response, and the cell cycle.

Methylation at mouse Cdkn1c is acquired during postimplantation development and functions to maintain imprinted expression.

Monoallelic expression of imprinted genes is generally associated with differential methylation. Methylation may be inherited as the gametic imprinting mark or may be acquired postfertilization. Here, we characterize a differentially methylated region associated with the mouse Cdkn1c gene and find that it is confined to a CpG island that begins 600 bp 5' of the promoter and extends into the transcription unit.