•Histone post-translational modifications (HPTMs) regulate gene transcription.•Specific proteins serve as “readers” of HPTMs.•Using HPTMs-containing peptides, proteins or nucleosomes to pull-down “readers”.•Using mass spectrometry to identify “readers”.The so-called “readers” of histone post-translational modifications (HPTMs) refer to proteins or complexes that are recruited to HPTMs thus eventually regulate gene transcription. To identify these “readers”, mass spectrometry plays an essential role following various enriching strategies. These enriching methods include the use of modified histone peptides/proteins or chemically synthesized histones/nucleosomes containing desired HPTMs to enrich the readers of HPTMs. Despite the peptide- or protein-based assay is straightforward and easy to perform for most labs, this strategy has limited applications for those weak or combinational interactions among various HPTMs and false-positive results are a potential big problem. While the results derived from synthesized histone proteins/nucleosomes is more reliable as it mimics the real chromatic conditions thus is able to analyze the binders of those cross-talked HPTMs, usually the synthesis is so difficult that their applications are impeded for high throughput analysis. In this review, an overview of these analytical techniques is provided and their advantages and disadvantages are discussed.

•We analyze histone modifications in two phenotypes of esophageal squamous cell carcinoma.•We evaluate the correlation between histone marks and cancer invasive capabilities.•A distinguished PTM, H4K79me2 was discovered and verified.Eukaryotic DNA is packaged into a chromatin with the help of four core histones (H2A, H2B, H3, and H4). Diverse histone post-translational modifications (PTMs) are hence involved in the regulation of gene transcription. However, how this regulation does work is still poorly understood and lacks details. Here we used the mass spectrometry-based proteomics approach to perform a comparative analysis of histone marks at a global level in two phenotypes of esophageal squamous cell carcinoma (ESCC) with different invasiveness. We obtained a comprehensive profiling of histone H3 and H4 PTMs including lysine methylation, acetylation and novel butyrylation. The correlation between histone marks and cancer invasive capabilities was further characterized and one distinguishable PTM, H4K79me2 was discovered and verified in this study. Immunohistochemistry analysis suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results enrich the dataset of the feature pattern of global histone PTMs in ESCC cell lines.Biological significanceCore histone proteins, decorated by multiple biological significant protein post-translational modifications (PTMs) such as lysine acetylation and lysine methylation, are considered to regulate gene transcription and be associated with the development of cancer. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers. However, the regulation mechanism is largely unknown. Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of esophageal squamous cell carcinoma (ESCC) cell lines with different invasive capabilities. To the best of our knowledge, it is the first report about the comparative analysis for histone marks of the different invasive ESCC cell lines. A significantly differential level of histone modification, H4K79me2, was determined and verified. Immunohistochemistry analysis further suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results could contribute to understanding the different expressions of global histone PTMs in different invasive ESCC cell lines.

Histone post-translational modifications (PTMs) have been considered to be a major group of important epigenetic marks and play critical roles in the regulation of chromatin-templated biological processes. To date, novel strategies for the quantification of histone PTMs are still highly desirable. Herein, we present an efficient approach to quantitatively characterize histone PTMs using stable isotope dimethyl labeling coupled with mass spectrometry. At first, all of the ε-amino groups of free lysines are derivatized by heavy formaldehyde to enable an easy distinction of free lysines from those of naturally occurring lysine-dimethylation upon MS analysis. After tryptic digestion, a second derivatization was applied with heavy- and light-stable isotope dimethyl labeling to label the N-termini of tryptic peptides from different sample sources. The mixture was further identified and quantified by HPLC-MS/MS. This method enables the comparison of histone PTMs from multiple sample sources and the quantification of different PTMs at certain amino-acid residues of histones in one single experiment. Thus, it is highly attractive for the identification of epigenetic histone marks.

Protein post-translational modifications (PTMs) play important roles in cellular physiology. Mass spectrometry (MS) has been developed into a powerful tool to identify all possible protein modifications. Herein, we describe our efforts to deduce the structures of two unknown modifications at tryptophan (Trp) residues (W + 92 Da and W + 108 Da). The two modifications were further confirmed by aligning the MS/MS fragmentation of synthetic peptide with in-vivo peptide identified. Finally, the mimic experiment elucidated how two Trp modifications occur. This study, therefore, expands current knowledge of Trp modifications.

•A vorinostat-based small molecule probe was designed, synthesized and immobilized onto the surface of sepharose beads.•Vorinosta-coated beads based proteomics approach for profiling of novel target proteins of vorinostat.•ENO-1 was detected and confirmed as a novel, potential target of vorinostat.Inhibitors of histone deacetylases (HDACs) have been considered to be new anticancer agents. As a key inhibitor of HDAC, vorinostat can cause growth arrest and death of a broad of transformed cells and interact with a variety of substrates. A comprehensive analysis of proteins interacting with HDAC inhibitors is of great importance in understanding molecular mechanisms of the drugs. Here, we reported the preparation and characterization of vorinostat-coated beads for profiling of novel target proteins of vorinostat (a key HDAC inhibitor). The enriched proteins were further analyzed by HPLC–MS/MS. Besides the known substrates, there were also several novel enriched protein candidates, one of which was a metalloenzyme α-enolase (ENO-1). According to our best knowledge, it is the first time that ENO-1 has been detected as a potential target of vorinostat through chemoproteomics approach. Further competition analysis indicated that ENO-1 may be co-enriched as a substrate complex. Our results demonstrated that the chemical probe combined with proteomics approach may be developed as a potential tool to identify target proteins of drugs.