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Enhanced top-down characterization of histone post-translational modifications

DOI: 10.1186/gb-2012-13-10-r86

Keywords: Saltless WCX-HILIC, top-down, histone, posttranslational modification

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Abstract:

Histones are important chromatin proteins that act as spools to package and order DNA into structural and manageable chromosomes. Core histones are modified by multiple post-translational modifications (PTMs) such as lysine acetylation, lysine or arginine methylation, and serine or threonine phosphorylation, among others. These PTMs generate a 'histone code' [1] that is implicated in chromatin-related cellular processes [2] including transcription [3], replication [4], repair [5], and alternative splicing [6].Although core histones comprise only four families (H4, H2B, H2A, and H3), each family has thousands of potential isoforms generated by different combinations of PTMs and protein sequence variation. Traditional antibody-based methods target specific isoforms, typically analyzing one PTM at a time, which makes it virtually impossible to measure combinatorial modifications occurring within the same histone molecule. Recently, high-throughput bottom-up [7] and middle-down [8] proteomic methods demonstrated potential for global characterization of PTMs on histone tails. However, these methods are ill-suited for characterizing multiple PTMs dispersed along the entire protein sequence that have been previously discovered to have significant participation in chromatin regulation [2,9-11].Top-down proteomic and high-throughput approaches are clearly required to identify and quantify the modulation of multiple intra-molecular histone modifications that synergistically regulate histone functions. Recently, a global top-down study demonstrated the feasibility of intact protein analysis for this purpose by identifying more than 300 histone isoforms using extensive fractionation and customized bioinformatics for global proteome characterization [12]. In histone-focused studies, top-down approaches using an offline two-dimensional liquid chromatography (2D LC) separation and Fourier transform mass spectrometry (FTMS) characterized 34 H4 isoforms from approximately 150 μg of

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