Histones are a class of highly conserved proteins with only a few amino acid differences between the sequences of different variants. Meanwhile, histone is a group of basic proteins that bind to DNA in eukaryotic nucleosomes with its N-terminal tail region containing a large number of post-translational modifications such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, and other modifications.
Dynamically regulated by modifying and de-modifying enzymes, histone modifications alter the electrical and hydrophobic properties as well as the spatial structure of amino acid residues, regulating the binding state of histones to DNA and affecting important biological processes such as DNA replication, DNA damage repair, and gene transcription. In addition, histone modifications can also recruit specific binding proteins and activate gene transcription factors relevant to physiological or pathological states. Without changes in DNA sequence, histone modifications can regulate gene expression and determine different phenotypes of organisms. Therefore, histone modifications are considered an important class of epigenetic code whose abnormalities are closely associated with the development of major diseases such as cancer. Currently, histone modification has become a hot spot for research on tumor markers and anti-tumor drugs.
The systematic identification of histone modifications is challenging due to the wide variety, diverse forms, and dynamic changes. In addition to the low binding protein abundance, histones also reserve weak binding power, which burdens effective identification in complex systems. Based on this, we have carried out a series of novel assays and techniques around the identification, enrichment, labeling, and characterization of histone modifications and their binding proteins. Moreover, we have also established a highly sensitive systematic analytical solution for histone modifications in combination with proteomics technology to study the dynamic changes of histone modifications in tumor cells, stem cells, and other cell lines in order to identify abnormal histone modifications in pathological states and explore new biological functions of histone modifications.
Creative Proteomics uses Thermo Fisher's Q ExactiveHF Mass Spectrometry platform, Orbitrap Fusion Mass Spectrometry platform, and Orbitrap Fusion Lumos Mass Spectrometry platform combined with Nano-LC to introduce a perfect solution for qualitative and quantitative analysis of histone modifications. By using this service, customers will only need to send in the sample of investigation, inform objectives of experiments and leave the rest to our team, including but not limited to the identification and quantification of histone modifications and modification sites.
Compared with traditional quantitative proteomic analysis, the key step in the quantitative analysis of histone modifications regards isolating and purifying the pure histone fractions. Meanwhile, histones are within the nucleus and have posed great isolation difficulties. To overcome this, Creative Proteomics has established an experimental platform to isolate and purify histones based on extensive references and an experienced technical team.
During histone isolation and purification, cell nuclei are first obtained via centrifugation, and then nuclei are destroyed to collect histones and DNA separately. Subsequently, impurities are sunk by the high-salt concentration loading buffer. As a result, the histone of various fractions is separated and purified by HPLC for subsequent mass spectrometry analysis. Meanwhile, in order to identify and quantify further histone post-translational modification information, Creative Proteomics utilizes 2-3 different digestive enzymes for protein sample digestion to maximize peptide coverage of histones in mass spectrometry and minimize the loss of histone post-translational modification information caused by inappropriate peptide length and low ionization efficiency.
Routes for the identification of post-translational modifications of histones
1. Tissue samples
Plant tissue samples: >400 mg
Blood samples: ≥2 mL (with EDTA for anticoagulation in plasma)
Serum: 2 mL
Urine: 10 mL
Animal tissue samples: ≥ 2 g
Cell samples: 1 x 10^8 cells
Yeast, microorganisms and others: dry weight 400 mg
2. Protein sample: total protein of 2-5 mg; common tissue and cell lysate can be used for protein extraction
3. Sample shipping: please use sufficient dry ice for shipping
1. Experimental procedures
2. Mass Spectrometry Parameters
3. Details of histone post-translational modifications
4. Mass Spectrogram