MacroH2A (mH2A) is one of the most recently identified members of the heteromorphous histone variant family. It is unique among the members of this group because it contains an unusually large non-histone C-terminal end, from where its name derives, and appears to be restricted to subphylum vertebrata. Although a concerted effort has been carried out in order to characterize the physiological relevance of mH2A, little is known in comparison about the structural importance of the molecule. Elucidating the biophysical and conformational proprieties of mH2A in chromatin may provide clues into the links between this histone variant and its unique function(s). In this paper, we look first at the heterogeneous tissue-specific distribution of this protein in different vertebrate classes. This is followed by a structural comparison between mH2A and H2A protein and by the characterization of the nucleosome core particles with which these histone subtypes are associated. We find that the highly alpha-helical C-terminus of mH2A confers an asymmetric conformation to nucleosomes and that this variant is tightly bound to chromatin fragments in a way that does not depend on the overall extent of acetylation of the other core histones.

BackgroundiTRAQ is a proteomics technique that uses isobaric tags for relative and absolute quantitation of tryptic peptides. In proteomics experiments, the detection and high confidence annotation of proteins and the significance of corresponding expression differences can depend on the quality and the species specificity of the tryptic peptide map database used for analysis of the data. For species for which finished genome sequence data are not available, identification of proteins relies on similarity to proteins from other species using comprehensive peptide map databases such as the MSDB.ResultsWe were interested in characterizing ripening initiation ('veraison') in grape berries at the protein level in order to better define the molecular control of this important process for grape growers and wine makers. We developed a bioinformatic pipeline for processing EST data in order to produce a predicted tryptic peptide database specifically targeted to the wine grape cultivar, Vitis vinifera cv. Cabernet Sauvignon, and lacking truncated N- and C-terminal fragments. By searching iTRAQ MS/MS data generated from berry exocarp and mesocarp samples at ripening initiation, we determined that implementation of the custom database afforded a large improvement in high confidence peptide annotation in comparison to the MSDB. We used iTRAQ MS/MS in conjunction with custom peptide db searches to quantitatively characterize several important pathway components for berry ripening previously described at the transcriptional level and confirmed expression patterns for these at the protein level.ConclusionWe determined that a predicted peptide database for MS/MS applications can be derived from EST data using advanced clustering and trimming approaches and successfully implemented for quantitative proteome profiling. Quantitative shotgun proteome profiling holds great promise for characterizing biological processes such as fruit ripening initiation and may be further improved by employing preparative techniques and/or analytical equipment that increase peptide detection sensitivity via a shotgun approach.