RESUMEN
DNA is organized into nucleosomes, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins including H2A, H2B, H3, and H4. Histones are critical regulators of many nuclear processes, including transcription, DNA damage repair, and higher order chromatin structure. Much of their function is mediated through extensive and dynamic posttranslational modification (PTM) by nuclear enzymes. Histone PTMs are thought to form a code, where combinations of PTMs are responsible for specific biological functions. Here, we present protocols to identify and quantify histone PTMs using nanoflow liquid chromatography coupled to mass spectrometry (MS). We first describe how to purify histones and prepare them for MS. We then describe three MS platforms for histone PTM analysis, including bottom-up, middle-down, and top-down approaches, and explain the relative benefits and pitfalls of each approach. We also include tips to increase the throughput of large experiments.
Asunto(s)
Histonas/química , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular/métodos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Histonas/genética , Histonas/aislamiento & purificación , HumanosRESUMEN
For the purpose of analytical investigation and structure/activity relationships, some secretin analogues and secretin fragments have been synthesized. HPLC comparison of the synthesized products with our synthetic secretin revealed about 2% [D-Ala17]secretin, 1% [D-Leu13]secretin and less than 1% aminoterminal degradation products. The D-Ala17 content can be eliminated if the starting material used for segment coupling (Z-Arg(Z2)-Asp(OBut)-Ser(But)-Ala-OH) has no D-Ala-contamination. In addition, traces of the rearrangement products [3-aspartoyl]-secretin and [beta-Asp3]secretin are suspected. Secretin can be degraded to several compounds by chromatography on a strong basic ion exchanger in 1% acetic acid. These products are more polar than secretin and have no biological activity. The secretin content measured by HPLC correlated well with the biological data, since the degradation products and other byproducts separated by HPLC have only a negligible influence on the pancreatic flow.