RESUMO
Histone acetylation and methylation play an important role in the regulation of gene expression. Irregular patterns of histone global acetylation and methylation have frequently been seen in various diseases. Quantitative analysis of these patterns is of high value for the evaluation of disease development and of outcomes from therapeutic treatment. Targeting histone acetylation and methylation by selected reaction monitoring (SRM) is one of the current quantitative methods. Here, we reported the use of the multiplexed parallel reaction monitoring (PRM) method on the QExactive mass spectrometer to target previously known lysine acetylation and methylation sites of histone H3 and H4 for the purpose of establishing precursor-product pairs for SRM. 55 modified peptides among which 29 were H3 K27/K36 modified peptides were detected from 24 targeted precursor ions included in the inclusion list. The identification was carried out directly from the trypsin digests of core histones that were separated without derivatization on a homemade capillary column packed with Waters YMC ODS-AQ reversed phase materials. Besides documenting the higher-energy c-trap dissociation (HCD) MS(2) spectra of previously known histone H3/H4 acetylated and methylated tryptic peptides, we identified novel H3 K18 methylation, H3 K27 monomethyl/acetyl duel modifications, H2B K23 acetylation, and H4 K20 acetylation in mammalian histones. The information gained from these experiments sets the foundation for quantification of histone modifications by targeted mass spectrometry methods directly from core histone samples.
Assuntos
Histonas/química , Acetilação , Lisina/química , Espectrometria de Massas , Metilação , Peptídeos/química , Propionatos/química , Tripsina/químicaRESUMO
OBJECTIVE: To establish a protocol for the targeting gene therapy against cancer with rich epidermal growth factor receptor (EGFR). METHODS: A recombinant pcDNA3.1-PE III mut was constructed and combined with a non-viral vector, a fusion protein histone H1, epidermal growth factor C-loop previously expressed by us, to be a protein-DNA complex in vitro. Using the complex to treat BT-325 and Hela cancer cells with EGFR and JK cells without EGFR. The killing rates of the cells was calculated after 48 h of incubation at 37 degrees C. RESULTS: To BT-325 and Hela cells, the killing rates were 46.03% and 48.12% respectively. To JK cells, the complex had no killing function. CONCLUSION: The protocol for targeting gene therapy against cancer with EGFR has been established successfully.
Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Receptores ErbB/genética , Exotoxinas/farmacologia , Marcação de Genes , Terapia Genética , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Virulência/farmacologia , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Sequência de Bases , Linhagem Celular Tumoral , Células , DNA/genética , Receptores ErbB/metabolismo , Exotoxinas/genética , Vetores Genéticos , Histonas/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
OBJECTIVE: To create a gene transfer vehicle for targeting gene therapy of cancer with epidermal growth factor receptor overexpressing. METHODS: Encoding sequences of the first domain of histone gene (H1) and EGF C-loop (EGFc) were obtained by PCR amplification. These two DNA fragments were ligated by EcoR I site, and cloned and sequenced. E. coli expression vector of the fusion gene was then constructed. The fusion protein H1EGFc was purified by specific band isolation from SDS-PAGE. RESULTS: The molecular weight of purified protein was consistent with the designed request. Its purity reached 94.02%. CONCLUSION: A fusion protein H1EGFc was expressed and purified.