Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography.
Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography.
复制标题
使用反相高效液相色谱法对泛素及其衍生物进行胰蛋白酶肽图分析。
DOI：
10.1016/0003-2697(86)90535-x
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发表时间：
1986
影响因子：
2.9
通讯作者：
Wilkinson,KD
中科院分区：
文献类型：
--
作者：
Cox,MJ;Shapira,R;Wilkinson,KD
胰蛋白酶消化和随后的肽映射的ATP依赖的蛋白水解辅因子泛素及其衍生物的条件进行了描述。在水溶液中，由76个氨基酸组成的天然泛素仅在精氨酸-74处发生单一切割。在6.5 M尿素中获得泛素的完全消化，尽管赖氨酸-33和精氨酸-74处的裂解缓慢。肽图通过反相高效液相色谱法与C18柱，使用三氟乙酸/三乙胺缓冲系统和乙腈作为洗脱剂来实现。使用线性梯度分离的肽通过氨基酸分析鉴定。通过该方法分析的衍生物包括氧化的、单碘酪氨酰和二碘酪氨酰泛素。这种技术将是有用的，在检查化学修饰的泛素肽的修饰程度和特异性。此外，该技术将有助于比较不同生物体的泛素肽。
The conditions for tryptic digestion and subsequent peptide mapping of the ATP-dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 m urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms.