{"title":"利用二维-TAU 凝胶电泳揭示组蛋白蛋白酶形式。","authors":"Marina La Chimia, Cristina Fontana, Arianna Cosentino, Chiara Bruno, Domenico Iacopetta, Domenica Scumaci","doi":"10.3791/67321","DOIUrl":null,"url":null,"abstract":"<p><p>Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2000,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis.\",\"authors\":\"Marina La Chimia, Cristina Fontana, Arianna Cosentino, Chiara Bruno, Domenico Iacopetta, Domenica Scumaci\",\"doi\":\"10.3791/67321\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.</p>\",\"PeriodicalId\":48787,\"journal\":{\"name\":\"Jove-Journal of Visualized Experiments\",\"volume\":\" 212\",\"pages\":\"\"},\"PeriodicalIF\":1.2000,\"publicationDate\":\"2024-10-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Jove-Journal of Visualized Experiments\",\"FirstCategoryId\":\"103\",\"ListUrlMain\":\"https://doi.org/10.3791/67321\",\"RegionNum\":4,\"RegionCategory\":\"综合性期刊\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Jove-Journal of Visualized Experiments","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.3791/67321","RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
引用次数: 0
摘要
组蛋白会发生各种翻译后修饰(PTM),如甲基化、乙酰化、磷酸化、酰化和泛素化,从而控制核小体的动态并决定细胞的命运。核小体是染色质的功能单位,由 DNA、构成球状核心的四对组蛋白(H3、H4、H2A 和 H2B)以及稳定染色质结构的连接组蛋白 H1 组成。组蛋白的氨基(N)末端尾部突出于球状核心结构域,并经历不同的 PTM,从而影响染色质结构。一些证据表明,组蛋白 PTM 的平衡对维持所有生理活动至关重要。组蛋白 PTM 的失调是细胞异常增殖、侵袭和转移的主要原因。因此,开发表征组蛋白 PTM 的方法至关重要。在这里,我们介绍了一种分离和分析组蛋白同工酶的有效技术。该方法基于两种正交分离的组合,可以富集组蛋白同工酶,并在随后进行质谱鉴定。该技术最初由 Shechter 等人描述,它结合了酸-尿素聚丙烯酰胺凝胶(TAU-GEL)和十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE),前者可根据大小和电荷分离碱性组蛋白,后者可根据分子量分离蛋白质。其结果是组蛋白异构体的二维图谱,适用于凝胶内消化,然后进行质谱鉴定和 Western 印迹分析。其结果是组蛋白同工酶的二维图谱,既适用于凝胶内消化,也适用于质谱鉴定和 Western 印迹分析。这种蛋白质组学方法是一种稳健的方法,可以富集单个组蛋白同工酶和表征新的组蛋白 PTM。
Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis.
Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.
期刊介绍:
JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.